Insect protein synthesis in frog cells: the translation of honey bee promelittin messenger RNA in Xenopus oocytes

Venom glands of young queen bees (Apis mellifera) synthesize the toxic peptide melittin as their main product. Melittin is formed by proteolytic cleavage of a precursor, promelittin. Unfractionated RNA prepared from venom glands was injected into Xenopus oocytes and was shown to direct the synthesis of a promelittin-like substance. About half of the peptide chain made in oocytes has been sequenced; the 17 amino acid residues identified correspond exactly with sequences found in promelittin from venom gland cells. These results yield final proof that injected messenger RNAs can be read with great fidelity. The translation of a messenger from an insect gland shows that at least some of the translational systems within the oocyte are neither cell-type nor phylum specific. It seems likely that the oocyte can be used to assay any kind of eukaryotic mRNA.The conversion of promelittin to melittin could not be detected in oocytes. Moreover, the promelittin synthesized in oocytes differs at the carboxyl end from the product made in gland cells, for the latter terminates with glutamine amide while the oocyte material probably ends with an amino acid with a free α-carboxyl group. Some of the post-translational modifications characteristic of gland cells thus do not seem to take place in oocytes.     

Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin.

Poly(A)-rich RNA isolated from ripening ovaries of snowdrop (Galanthus nivalis L.) yielded a single 17-kDa lectin polypeptide upon translation in a wheat-germ cell-free system. This lectin was purified by affinity chromatography. Translation of the same RNA in Xenopus leavis oocytes revealed a lectin polypeptide which was about 2 kDa smaller than the in vitro synthesized precursor, suggesting that the oocyte system had removed a 2-kDa signal peptide. A second post-translational processing step was likely to be involved since both the in vivo precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide. This hypothesis was confirmed by the structural analysis of the amino acid sequence of the mature protein and the cloned mRNA. Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification, allowed determination of the complete 105 amino acid sequence of the snowdrop lectin polypeptide. Comparison of this sequence with the deduced amino acid sequence of a lectin cDNA clone revealed that besides the mature lectin polypeptide, the lectin mRNA also encoded a 23 amino acid signal-sequence and a C-terminal extension of 29 amino acids, which confirms the results from in vitro translation experiments.     

Structure of a precursor to human pancreatic polypeptide

We have isolated mRNA from a human pancreatic islet cell tumor and have identified among the cell-free translation products a precursor of pancreatic polypeptide with an approximate Mr = 11,000. Recombinant DNA molecules encoding this precursor were selected from a cDNA library prepared from the islet tumor mRNA. From the nucleotide sequences of cDNAs encoding the precursor, we have deduced the complete amino acid sequence of pre-propancreatic polypeptide. These sequences encode a protein consisting of 95 amino acid residues with a Mr = 10,432. The sequence of human pancreatic polypeptide occurs in the middle of the precursor and is flanked at its carboxyl terminus by a 27-amino acid sequence which is similar to a peptide previously isolated from canine pancreatic islets. At the amino terminus of the precursor is a probable leader sequence which is rich in hydrophobic residues. A smaller pancreatic polypeptide-related protein was generated in cell-free translations of mRNA supplemented with microsomal membranes. Sequential Edman degradations of this smaller peptide indicate that the sequence of pancreatic polypeptide is located at the amino terminus of the prohormone.     

Solid-phase synthesis of melittin: purification and functional characterization

The main component of the honey bee venom, melittin, is a cationic polypeptide containing 26 amino acids. Exposure of lipid bilayers to this peptide results in the formation of anion-selective channels with a variety of unit conductances. One of the possible causes for this heterogeneity in the conductance could be heterogeneity of the melittin preparation, and indeed, the existence of two prominent forms of naturally occurring melittin, differing only at the N-terminal amino group, has been documented. This paper describes the synthesis of the major form of melittin, using stepwise solid-phase methodology and the demonstration that the synthetic melittin, devoid of the minor component (N-formylmelittin) and other contaminants, interacts with lipid bilayers to form channels which are qualitatively indistinguishable from the ones formed by the naturally occurring toxin. This result indicates that the heterogeneity in the channels produced in bilayers by bee venom is not due to differences in the channel-forming properties of the formyl and non-formyl melittin but rather to differences in the number and orientation of melittin monomers of identical primary structure as they aggregate to form channels in the lipid bilayer.     

Mosquito vitellogenin subunits originate from a common precursor

Using a cell-free translation system, we demonstrated that the two subunits of mosquito vitellogenin (VG), 200 kDa and 65 kDa, originate from a common precursor. The precursor polypeptide of 220 kDa is a translation product specific to mRNA from vitellogenic mosquitoes. In immunoprecipitation analysis, the 220-kDa polypeptide was recognized by monoclonal antibodies directed either to the large or the small VG subunit. Peptide mapping showed homology between the 220-kDa polypeptide and both subunits, thus providing further proof that the 220-kDa product of translation is the precursor for both VG subunits. In the presence of microsomal membranes, the molecular size of the VG precursor increased to 235 kDa suggesting this as a first step in co-translational modifications of VG.     

Isolation and cell-free translation of a human immunoglobulin light chain messenger RNA.

Human myeloma cell line RPMI 8226 synthesizes and secretes a lambda immunoglobulin light chain. Total cellular RNA, obtained from cells grown in culture, was used for the isolation of poly(A)-containing mRNA by oligo(dT)-cellulose chromatography. The poly(A)-containing mRNA was translated in an Ehrlich ascites extract. Immunoprecipitation of the cell-free products showed that a polypeptide chain antigenically related to human lambda chain was synthesized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the cell-free product was larger than the secreted light chain. On tryptic digestion the cell-free product and the secreted light chain exhibited essentially identical peptide patterns except for an additional peptide derived from the cell-free product. We conclude that the lambda mRNA from this human myeloma cell line codes for a precursor to the secreted lambda chain as described for immunoglobulin mRNAs from murine plasmacytomas.     

In vitro synthesis of bean (Phaseolus vulgaris) chloroplastic and cytoplasmic leucyl-tRNA synthetases. Characterization and processing of a precursor polypeptide for the chloroplast enzyme

Bean (Phaseolus vulgaris) chloroplastic and cytoplasmic leucyl-tRNA synthetases differ in their structural and catalytic properties and do not share common antigenic determinants. Polyadenylated mRNAs, prepared from young bean leaves, have been translated in vitro in a rabbit reticulocyte lysate cell-free system. The newly synthesized polypeptides have been submitted to immunoadsorption on protein A-Sepharose in the presence of the antibodies raised against the chloroplastic or the cytoplasmic leucyl-tRNA synthetase. The specificity of the immunoadsorption has been checked by competition experiments involving the pure enzymes. Bean chloroplastic leucyl-tRNA synthetase is synthesized in vitro from a polyadenylated mRNA as a precursor polypeptide of 130 kDa, which is somewhat larger than the mature enzyme of 120 kDa. Bean cytoplasmic leucyl-tRNA synthetase is synthesized in vitro as a polypeptide which has the size of the mature monomer (130 kDa). Processing of the precursor polypeptide of the chloroplastic leucyl-tRNA synthetase, yielding the mature enzyme, has been obtained by performing the in vitro translation in the presence of canine pancreatic microsomal membranes. These results suggest that in vivo bean chloroplastic leucyl-tRNA synthetase could be synthesized in the cytoplasm as a precursor which would be transported into the chloroplasts.     

蜂毒溶血肽(melittin)cDNA在大肠杆菌高表达质粒中的克隆(英文)

本项研究从新羽化的蜜蜂蜂王毒腺中提取了总mRNA,用逆转录的方法,合成了cDNA,并将其克隆到了噬菌体质粒λgtll的EcoRI位点,建立了melittin的cDNA文库。用PCR扩增技术从cDNA文库中产生了长度为87bp的melittin基因,并将其插入到高表达载体pBV220的EcoRI和pstI位点构成重组质粒PBM95,并转化到大肠杆菌JM101的感受态细胞中。经过在含氨苄青霉素的LB平板上对转化子进行筛选和对来自转化子中的重组质粒PBM95的酶切分析及melittin基因的测序,证明melit－tincDNA克隆成功。     

Identification of a viral protein involved in post-translational maturation of the encephalomyocarditis virus capsid precursor.

Translation of encephalomyocarditis virus RNA in a cell-free system from uninfected Krebs ascites cells results in the synthesis of a major polypeptide product with a molecular weight of approximately 112,000. In contrast, when the viral RNA is translated in a cell-free system from virus-infected cells, this polypeptide is absent and the largest polypeptide produced has a molecular weight of about 100,000. This latter polypeptide comigrates on sodium dodecyl sulfate-gels with in vivo virus capsid precursor A, and the two have identical patterns of CNBr-generated peptides. A polypeptide having a molecular weight of 12,500 is also a major translation product in the system from infected cells (but not from uninfected cells). This polypeptide appears to be generated by cleavage of the NH-2-terminal portion of the viral RNA-dependent polypeptides by a proteolytic activity present in the infected cell-free system. This proteolytic activity copurifies with the 23,000-molecular weight viral capsid protein gamma, found in infected cells, through chromatography on DEAE-cellulose and cellulose phosphate. This suggests that gamma is itself a proteolytic enzyme involved in maturation of the viral capsid precursor.     

One amino acid change in rat SMR1 polypeptide induces a 1 kDa difference in its apparent molecular mass determined by electrophoretic analysis

SMR1 is a male-specific, 19 kDa, in vitro translation product of Wistar rat submaxillary glands, which may be the precursor of a small hormone resembling the TRH. In Sprague-Dawley and Fischer rats, instead of SMR1, a male-specific 18 kDa polypeptide may be found. We have cloned the cDNA encoding the 18 kDa polypeptide. We show that the 19 and the 18 kDa polypeptides have the same sequence except for one amino and change.     

CLONING OF MELITTIN cDNA FROM HONEYBEE INTO THE HIGH EXPREHION PLASMID OF ESCHERICHIA COLI*

Abstract Total mRNA from venom glands of newly emerged queen bees was reversely transcribed into cDNA and cloned into the EcoRI site of plasmid λgt11; cDNA library for bee venom was thus constructed. PCR technique was used to produce the melittin coding sequence from the cDNA library. A 87 bp product was produced and inserted into the EcoRI and PstI sites of the high level expression vector pBV220. Recombinant plasmid pBM95 was transformed into the competent cells of E.coli JM101. After screening transformants on LB medium with ampicilin, structure of the recombinant plasmid pBM95 from transformants was analyzed and melittin gene in pBM95 was sequenced. The cloned cDNA coding for honey bee melittin was obtained.     

The sting. Melittin forms channels in lipid bilayers

Melittin, a toxin of bee venom, is a cationic polypeptide composed of 26 amino acids. The six residues of the C-terminal end are polar and 19 of the 20 residues of the N-terminal end are hydrophobic. Exposure of the lecithin bilayer to melittin results in the formation of channels that are more permeable to anions that to cations. Unilateral addition of melittin produces a voltage-dependent increase in membrane conductance when the side where the polypeptide is present in made positive but not when it is made negative. At a fixed voltage, the conductance increases with the fourth power of the melittin concentration in the aqueous phase. At a fixed peptide concentration, the conductance increases approximately e-fold per 6-mV increase in the electrical potential difference across the membrane. These results suggest that four melittin monomers are needed to form a channel and, furthermore, that a minimum of four equivalent electronic charges need to be displaced by the electrical field to explain the voltage dependence of the conductance.     

Identification of N-terminal methionine in the precursor of immunoglobulin light chain. Initiation of translation of messenger ribonucleic acid in plants and animals.

The proteins programmed in the wheat-germ cell-free system by the mRNA coding for the MOPC-321 mouse myeloma L (light) chain were labelled with [35S]methionine, [4,5-3H]leucine or [3-3H]serine, and were subjected to amino acid-sequence analyses. Over 95% of the total cell-free product was sequenced as one homogeneous protein, which corresponds to the precursor of the L-chain protein. In the precursor, 20 amino acid residues precede the N-terminus of the mature protein. This extra piece contains one methionine residue at the N-terminus, one serine residue at position 18, and six leucine residues, which are clustered in two triplets at positions 6, 7, 8 and 11, 12, 13. The identification of methionine at the N-terminus of the precursor is in agreement with the evidence showing that unblocked methionine is the initiator residue for protein synthesis in eukaryotes. The absence of methionine at position 20, which precedes the N-terminal residue of the mature protein, suggests that myeloma cells synthesize the precursor. However, within the cell the precursor should be rapidly processed to the mature L chain, since precursor molecules have not yet been found in the intact animal. The abundance (30%) of leucine residues indicates that the extra-piece moiety is quite hydrophobic. The extra piece of the MOPC-321 L-chain precursor synthesized with the aid of the Krebs II ascites cell-free system is of identical size and it has the same leucine sequence [Schechter et al. (1975) Science 188, 160-162]. This indicates that cell-free systems derived from the plant and animal kingdom initiate mRNA translation from the same point. It is shown that the amino acid sequence of minute amounts of a highly labelled protein (0.1 pmol) can be faithfully determined in the presence of a large excess (over 2000 000-fold) of unrelated non-radioactive proteins.     

Purification from bee venom of melittin devoid of phospholipase A2 contamination

Conditions for the inactivation of phospholipase A₂ which contaminates melittin preparations were studied. A method for the purification of that peptide from bee venom is proposed. It gives, with a high recovery, a product devoid of phospholipase A₂ activity. In the first step, the venom is fractionated by gel filtration. Then the phospholipase A₂ still present in the melittin fraction is destroyed by sequential sulfitolysis and cyanogen bromide cleavage. This leaves the melittin intact. The final cation-exchange chromatography yields an homogeneous melittin preparation as analyzed by gel filtration, reverse-phase HPLC, and amino acid analysis.     

Cell-free synthesis of rat parotid preamylase.

Poly(A)-containing RNA from rat parotid gland directs the cell-free synthesis of several products in the reticulocyte lysate translation system including a very prominent 58,000-dalton polypeptide which is immunoreactive with anti-alpha-amylase. Purified alpha-amylase has a molecular weight estimated as 56,000 daltons. The 58,000-dalton, cell-free product and alpha-amylase share common peptides as determined by analysis of their limited proteolysis digests. The cross-reactivity and peptide homology suggest that the cell-free product may be a precursor of mature alpha-amylase. While the NH2 terminus of alpha-amylase is blocked, that of the 58,000-dalton product evidently is not, and automated sequence analysis has yielded its partial sequence as: Met-X-Phe-Phe-Leu-Leu-Leu-X-Leu-Ile-X-Leu-X-X-X-X-X-X-X-X-X-Phe-X-X-X-X-X-Ile-X-X-Leu-Phe. The highly hydrophobic nature of the NH2 terminus of the 58,000-dalton, cell-free product suggests that, like other secreted polypeptides, the extra piece may play a role in the transport and secretion of the mature alpha-amylase.     

Amino acid sequences which promote and prevent the binding and membrane insertion of surface-active peptides: comparison of melittin and promelittin

The temporal sequence of molecular events involved in the interactions of a number of related peptides with membranes are revealed using two complementary fluorescence techniques. Comparative studies are reported of the interactions of melittin, promelittin and a melittin analogue with trp-19 replaced with Ile and the n-terminal gly replaced with a trp residue, with phosphatidylcholine membranes. It is shown that the interaction of the n-terminal region of melittin rapidly binds and inserts into the body of the membrane with a rate constant of around 367 s-1. This is followed by a slightly slower membrane insertion of the trp-19 region with a rate constant of around 112 s-1. The positive charges of the melittin molecule then come into close proximity with the membrane with rate constants around 27 s-1. Finally, these charged regions insert into the hydrophobic core of the membrane with rate constants of about 0.3 s-1. The effect of incorporating net negative charge onto the membrane surface in the form of 15 mole % phosphatidylserine, augments by about threefold, the binding of the charged domains of the melittin molecule. The observations of the melittin interactions are compared with the melittin-precursor protein, promelittin. Sections of the promelittin molecule are also found to bind and insert into the body of the phospholipid membrane, although nearly 30 times less rapidly than melittin. No charged sections of promelittin are found to insert into the membrane.     

Rabbit C-reactive protein. Biosynthesis and characterization of cDNA clones

To study the biosynthesis of rabbit C-reactive protein (CRP), a cDNA library was constructed from CRP mRNA-enriched polysomal poly(A) RNA. Four recombinant plasmids, designated pCX9, pCX23, pCX28, and pCX39, from 39 positive clones were sequenced and found to represent overlapping clones. DNA sequencing of CRP cDNA and primer extension of the 5'-end of CRP mRNA have demonstrated that the complete length of rabbit CRP mRNA consists of 2331 nucleotides and a terminal poly(A) segment. Analysis of the resulting sequence indicated that rabbit CRP mRNA contained a 5'-noncoding region of 107 nucleotides, a leader sequence encoding 20 amino acids, a coding region covering 205 amino acids, and a 3'-noncoding region of 1549 nucleotides. The 3'-noncoding region contained a consensus AAUAAA sequence that is 105 nucleotides upstream from the 3'-terminal poly(A) segment. Using an in vitro translation system, we have confirmed that CRP is synthesized as a precursor polypeptide (Mr approximately equal to 26,000) which undergoes processing to form the mature polypeptide (Mr approximately equal to 23,500). The CRP precursor failed to display a calcium-dependent affinity for phosphorylcholine ligand as demonstrated by mature CRP, suggesting that the phosphorylcholine-binding site of CRP only formed after processing. Northern blot analysis suggested that following induction with turpentine, liver was the only site where CRP mRNA synthesis could be demonstrated and that the change in mRNA concentration correlated with the course of CRP production. Southern blot analysis of liver genomic DNA indicated a single gene copy for CRP.