Rabbit Reticulocyte Translation System Research Articles

YRDC Mediates the Resistance of Lenvatinib in Hepatocarcinoma Cells via Modulating the Translation of KRAS.

Lenvatinib is the latest and promising agent that has demonstrated a significant improvement of progression-free survival in advanced hepatocellular carcinoma (HCC). However, resistance emerges soon after initial treatment, limiting the clinical benefits of lenvatinib. Therefore, understanding the mechanism of resistance is necessary for improving lenvatinib efficacy. YRDC promotes the proliferation of hepatocarcinoma cells via regulating the activity of the RAS/RAF/MEK/ERK pathway, which was the primary pathway of the anticancer effect of lenvatinib. The purpose of this study is to investigate whether YRDC modulates the sensitivity of lenvatinib in hepatocarcinoma cells. Using the CCK-8 cell viability assay, wound-healing assay and clone formation assay in cell models, and xenograft assay in null mouse, we demonstrated that Huh7 cells with YRDC knockdown showed decreased susceptibility to lenvatinib than their control cells. Furthermore, we found that lenvatinib inhibited the expression of YRDC in a time-dependent manner. This effect may aggravate resistance to lenvatinib in hepatocarcinoma cells and may be an underlying cause of resistance, which emerges soon after lenvatinib initial treatment. To investigate how YRDC modulates the sensitivity of lenvatinib, we assessed the effect of tRNA with different t6A levels on the translation of the KRAS gene by in vitro rabbit reticulocyte translation system and measured the expression levels of the KRAS gene by western blot together with qPCR. We found that YRDC regulates the protein translation of KRAS in cell models, and the tRNA with low t6A modification level reduces the translation of the KRAS in the in vitro translation system. These results suggested that YRDC mediates the resistance of lenvatinib in hepatocarcinoma cells via modulating the translation of the KRAS. In this study, YRDC was confirmed to be a potential novel predictive biomarker of lenvatinib sensitivity in HCC.

Abstract B19: Functional translation of total RNA packaged in microparticles shed from multidrug resistant cancer cells

Abstract Microparticles (MPs) are submicron (0.1 -1 µM) membrane vesicles released from various cell types including malignant cells. Long considered to be inert cellular debris, MPs have recently been identified to play a crucial role in cellular cross-talk. MPs represent a heterogeneous mixture of surface antigens, proteins, lipids and cytoplasmic components resembling its parental cell. Our previous studies have identified the packaging and transfer of the transcripts, regulatory nucleic acids and proteins which are associated with the acquirement of multidrug resistance (MDR) between cancer cells. In this study we verify the functionality of packaged transcripts via in vitro translation of extracted total RNA from MPs isolated from cancer cells. We confirm the translation of exogenous RNA in a rabbit reticulocyte translation system via Western Blot analysis, 2-D SDS and mass spectrometry. These findings have significance in understanding the impact of MPs on cancer cell biology and reiterate their potential as a target in clinical oncology. Citation Format: Jamie Fung Lu, Deep Pokharel, Frederick Luk, Mary Bebawy. Functional translation of total RNA packaged in microparticles shed from multidrug resistant cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B19.

Polycystin-1 Protein Level Determines Activity of the Gα12/JNK Apoptosis Pathway

Mutations in PKD1 are the most common cause of autosomal dominant polycystic kidney disease (ADPKD). The protein product of PKD1 (polycystin-1 (PC1)) is a large transmembrane protein with a short intracellular C terminus that interacts with numerous signaling molecules, including Galpha(12). Cyst formation in ADPKD results from numerous cellular defects, including abnormal cilia, changes in polarity, and dysregulated apoptosis and proliferation. Recently, we reported increased apoptosis in Madin-Darby canine kidney (MDCK) cells through Galpha(12) stimulation of JNK and degradation of the anti-apoptotic protein Bcl-2 (Yanamadala, V., Negoro, H., Gunaratnam, L., Kong, T., and Denker, B. M. (2007) J. Biol. Chem. 282, 24352-24363). Herein, we confirm this pathway in Galpha(12)-silenced MDCK cells and utilize MDCK cell lines harboring either overexpressed or silenced PC1 to demonstrate that PC1 expression levels determine activity of the JNK/Bcl-2 apoptosis pathway. PC1-overexpressing MDCK cells were resistant to thrombin/Galpha(12)-stimulated apoptosis, JNK activation, and Bcl-2 degradation. In contrast, PC1-silenced MDCK cells displayed enhanced thrombin-induced apoptosis, JNK activity, and Bcl-2 degradation. In pulldown experiments, PC1 bound to Galpha(12), but not the related Galpha(13) subunit, and thrombin-stimulated MDCK cells led to increased interaction of Galpha(12) with the PC1 C terminus. In transient transfection assays, a PC1 C-terminal mutant lacking the G protein-binding domain was uncoupled from PC1-inhibited apoptosis. PC1 expression levels may be increased or decreased in ADPKD, and these findings suggest a mechanism in which levels of PC1 expression modulate Galpha(12)/JNK-stimulated apoptosis. Taken together, these findings are consistent with a set point model in which PC1 expression levels regulate specific G protein signaling pathways important to cyst development.

24] Selecting and evolving functional proteins in vitro by ribosome display

Publisher Summary In vivo systems have a number of limitations. One important restriction is that the library size is limited by the transformation efficiency, a step that all of these techniques have in common. Another limitation of in vivo -based selection methods becomes apparent if a diversification step must be included. It requires either repeatedly switching between in vivo selection and in vitro diversification or the use of mutator strains. The former approach is quite laborious, as it makes a new library generation and large-scale transformation necessary for each cycle of sequence diversification and selection. The latter approach may have the disadvantage that possible candidate molecules may be removed from the library by mutations generated either in the host genome or in the plasmid regions important for expression or replication. All these limitations can be simultaneously overcome in ribosome display. Ribosome display is an in vitro technology for the simultaneous selection and evolution of proteins from diverse libraries. Because no transformation is necessary, large libraries can be prepared and applied for selection. Diversification is conveniently introduced in this method, thereby making evolutionary approaches easily accessible. Ribosome display was first applied to short peptides and has subsequently been improved to work with folded proteins. This chapter describes in detail the methodology and the nature of the improvements and provides the information necessary to perform ribosome display for the selection and evolution of functional proteins, using either an E. coli or a rabbit reticulocyte translation system.

The herpes simplex virus vhs protein induces endoribonucleolytic cleavage of target RNAs in cell extracts.

The herpes simplex virus virion host shutoff (vhs) protein (UL41 gene product) is a component of the HSV virion tegument that triggers shutoff of host protein synthesis and accelerated mRNA degradation during the early stages of HSV infection. Previous studies have demonstrated that extracts from HSV-infected cells and partially purified HSV virions display vhs-dependent RNase activity and that vhs is sufficient to trigger accelerated RNA degradation when expressed as the only HSV protein in an in vitro translation system derived from rabbit reticulocytes. We have used the rabbit reticulocyte translation system to characterize the mode of vhs-induced RNA decay in more detail. We report here that vhs-dependent RNA decay proceeds through endoribonucleolytic cleavage, is not affected by the presence of a 5' cap or a 3' poly(A) tail in the RNA substrate, requires Mg(2+), and occurs in the absence of ribosomes. Intriguingly, sites of preferential initial cleavage were clustered over the 5' quadrant of one RNA substrate that was characterized in detail. The vhs homologue of pseudorabies virus also induced accelerated RNA decay in this in vitro system.

Inefficient reinitiation is responsible for upstream open reading frame-mediated translational repression of the maize R gene.

Maize R genes encode a small family of transcriptional activators of several structural genes in the anthocyanin biosynthetic pathway. The 5' leader region of most R genes contains a 38-codon upstream open reading frame (uORF) that previously was shown to be responsible for the repression of downstream gene expression in a transient transformation assay. In this study, we report that the 5' leader also can repress translation of the downstream luciferase gene both in the rabbit reticulocyte translation system and in transgenic rice plants. The ability to visualize the uORF peptide after in vitro translation permits quantification of both products of dicistronic mRNAs. Similarly, the construction of transgenic rice plants expressing wild-type and mutant constructs permits the quantification and correlation of steady state mRNA levels and reporter gene activities. Using these assays, we demonstrate directly that translation of the uORF is required for repression, that increasing translation of the uORF peptide decreases downstream gene expression, and that repression is unaffected by either subtle or gross changes in the uORF peptide. Rather, we find that ribosomes that translate the uORF reinitiate inefficiently and that the intercistronic sequence downstream of the uORF mediates this effect.

Trichothecene 3-O-Acetyltransferase Protects Both the Producing Organism and Transformed Yeast from Related Mycotoxins: CLONING AND CHARACTERIZATION OF Tri101

Trichothecene mycotoxins such as deoxynivalenol, 4,15-diacetoxyscirpenol, and T-2 toxin, are potent protein synthesis inhibitors for eukaryotic organisms. The 3-O-acetyl derivatives of these toxins were shown to reduce their in vitro activity significantly as assessed by assays using a rabbit reticulocyte translation system. The results suggested that the introduction of an O-acetyl group at the C-3 position in the biosynthetic pathway works as a resistance mechanism for Fusarium species that produce t-type trichothecenes (trichothecenes synthesized via the precursor trichotriol). A gene responsible for the 3-O-acetylation reaction, Tri101, has been successfully cloned from a Fusarium graminearum cDNA library that was designed to be expressed in Schizosaccharomyces pombe. Fission yeast transformants were selected for their ability to grow in the presence of T-2 toxin, and this strategy allowed isolation of 25 resistant clones, all of which contained a cDNA for Tri101. This is the first drug-inactivating O-acetyltransferase gene derived from antibiotic-producing organisms. The open reading frame of Tri101 codes for a polypeptide of 451 amino acid residues, which shows no similarity to any other proteins reported so far. TRI101 from recombinant Escherichia coli catalyzes O-acetylation of the trichothecene ring specifically at the C-3 position in an acetyl-CoA-dependent manner. By using the Tri101 cDNA as a probe, two least overlapping cosmid clones that cover a region of 70 kilobase pairs have been isolated from the genome of F. graminearum. Other trichothecene biosynthetic genes, Tri4, Tri5, and Tri6, were not clustered in the region covered by these cosmid clones. These new cosmid clones are considered to be located in other parts of the large biosynthetic gene cluster and might be useful for the study of trichothecene biosynthesis.

High-Level Expression of Human Thymidylate Synthase

A method is presented for expressing human thymidylate synthase (TS) to the extent of 25–30% of the protein inEscherichia coli.By this procedure, 200–400 mg of pure enzyme can be obtained from a 2-liter culture of cells. The key to the level of expression appears to be related to the conversion of purine bases in the third, fourth, and fifth codons of the TS cDNA to thymine, without altering the encoded protein product. Conversion of the penultimate proline to a leucine did not diminish expression, but while the isolated native enzyme contained only proline on its amino-terminal end, the mutated enzyme was found to contain methionine on its amino terminus. By contrast, the expression of the unmodified TS cDNA represented only about 0.1–0.2% of the total cellular protein. Unlike recombinant rat and human TSs, the respective enzymes purified to homogeneity from eukaryotic cells were blocked at the amino ends and possessed 2- to 4-fold lower specific activities. To determine at what level the impairment of expression occurred, anin vitrotranscription, translation system was employed and the results showed that while transcription was unaffected, the translation of native TS mRNA was reduced by at least 20-fold relative to modified TS mRNA using a rabbit reticulocyte translation system. Thus, it appears that at least for the TS gene, expression is greatly influenced by the GC content of the 5′ coding region of the gene in both prokaryote and eukaryote systems.

Overexpression of pokeweed antiviral protein in Escherichia coli: purification, characterization and comparison with native sources

Pokeweed antiviral proteins (PAP, PAP-II, PAP-S) represent a family of ribosome inactivating proteins (RIPs) isolated from various organs and at different stages of development of Phytolacca americana (pokeweed). They not only inhibit the mechanical transmission of plant viruses but also inactivate both eukaryotic and prokaryotic protein synthesis. One of them, PAP, was expressed with the help of the highly inducible prokaryotic expression vector pOPE 90 in Escherichia coli and the biological activity of the recombinant PAP (rPAP) was compared with the PAP purified from spring leaves of Phytolacca americana. After extraction and solubilization, rPAP was highly purified by anion and cation exchange chromatography. Immunoblotting with antibodies revealed identical immunological reactivity between recombinant or native PAP. In a rabbit reticulocyte translation system, rPAP was more efficient than native PAP to inhibit protein synthesis. The values of IC 50 from native PAP and recombinant PAP were determined as 5.10 −11 M and 5.10 −12 M, respectively. No difference was observed when the toxicity of native and rPAP was tested on established cell lines. PAP can be expressed in equivalent active form in Escherichia coli and its properties were thus similar to those of the native protein. Post-translational modifications of PAP in pokeweed did not seem to alter its ability to inhibit protein synthesis.

Formation of a Ligand-binding Site for the Acetylcholine Receptor in Vitro

Investigation of the mechanisms by which the subunits of ligand-gated ion channels fold and associate to form oligomers has been hampered by the lack of an in vitro system in which these reactions occur. We have established conditions in a rabbit reticulocyte translation system supplemented with canine pancreatic microsomes under which the alpha and delta subunits of the nicotinic acetylcholine receptor (AChR) fold and assemble to form a heterodimer with a cholinergic binding site comparable with that found in the intact AChR. Folding of the alpha subunit was followed by its ability to bind alpha-bungarotoxin. Folding efficiency was highly sensitive to changes in the redox potential of the translation medium and was favored by an oxidizing environment. Acquisition of the toxin binding conformation required N-linked core glycosylation but not oligosaccharide trimming, suggesting that oligosaccharide-dependent interaction of chaperones with the alpha subunit is not essential for correct subunit folding. The conformationally mature alpha subunit specifically associated with the delta subunit but not the beta subunit to form a heterodimer with a high affinity ligand-binding site. These data demonstrate, for the first time, correct folding and assembly of the AChR subunits in an in vitro system.

Internal 6-methyladenine residues increase the in vitro translation efficiency of dihydrofolate reductase messenger RNA

N 6-Methyladenosine (m6A) is found internally in a number of mRNA molecules from higher eucaryotic cells. In these investigations, it was found that the presence of m6A residues increase the in vitro translation efficiency of capped T7 transcripts of mouse dihydrofolate reductase (DHFR) mRNA. Using an in vitro rabbit reticulocyte translation system, the formation of internal m6A residues in the DHFR transcripts resulted in a 1.5-fold increase in translated DHFR compared to transcripts void of internal m6A residues. Translation in a wheat germ system, however, resulted in no increase in translation efficiency upon m6A formation, suggesting that the mechanism may be species-specific.

The Glut 1 Glucose Transporter Interacts with Calnexin and Calreticulin

Calnexin is an integral membrane protein that acts as a chaperone during glycoprotein folding in the endoplasmic reticulum. Cross-linking studies were carried out with the aim of investigating the interactions of calnexin with glycoproteins in vitro. A truncated version of the integral membrane glycoprotein Glut 1 (GT155) was synthesized in a rabbit reticulocyte translation system in the presence of canine pancreatic microsomes. Following immunoprecipitation with an anticalnexin antiserum, a cross-linker-independent association was observed between GT155 and calnexin. In addition, the anti-calnexin antiserum immunoprecipitated a UV-dependent cross-linking product consisting of GT155 and a protein of approximately 60 kDa designated CAP-60 (calnexin-associated protein of 60 kDa). Both the GT155-calnexin and the GT155-CAP-60 interactions were dependent on the presence of a correctly modified oligosaccharide group on GT155, a characteristic of many calnexin interactions. A GT155 mutant that was not glycosylated (AGGT155) did not associate with calnexin or CAP-60. Calreticulin, the soluble homologue of calnexin, was also shown to interact with GT155 only when the protein bore a correctly modified oligosaccharide group. Thus, our data show that both calnexin and calreticulin with Glut 1 in a glycosylation-dependent manner.

In vitro translation and membrane topology of rat recombinant mGluR 1α

The structure and post-translational processing of the metabotropic glutamate receptor 1α (mGluR1α) was analysed by in vitro cell-free translation, protease protection and deglycosylation. We show that mGluR1α can be synthesized in the rabbit-reticulocyte translation system to yield a predominant polypeptide product with an apparent molecular weight of 142 kDa. In the presence of dog-pancreatic microsomes this polypeptide was processed to an apparent molecular weight of 147 kDa. Treatment with the enzyme peptide- N-glycosidase F (PNGF) demonstrated that the increase in the apparent molecular weight of the processed translation product was due to N-linked glycosylation. Addition of the non-selective protease, proteinase K, resulted in the loss of this 147 kDa band and the appearance of a protected fragment of approx 92 kDa. A carboxy-terminal deletion mutant of mGluR1α was almost completely protected from protease action. These data show that the amino terminal of mGluR1α is translocated into the lumen of the endoplasmic reticulum and will consequently be located extracellularly when targeted to the plasma membrane. The data presented here on mGluR1α indicates the potential of in vitro translation and protease protection in the study of the molecular structure and processing of glutamate receptors.

Isolation and characterization of a cDNA clone encoding the pokeweed antiviral protein II from Phytolacca americana and its expression in E. coli

Three distinct ribosome-inactivating proteins (RIPs) were isolated from pokeweed Phytolacca americana We identified and sequenced for the first time a complete cDNA encoding the pokeweed antiviral protein II (PAP II), which is expressed in the late summer leaves of pokeweed. The cDNA of PAP II consists of 1, 187 nucleotides and encodes a mature protein of 285 amino acids. Its predicted amino acid sequence is only 33% similar to PAP and PAP-S. The NH 2 terminal extrapeptide (25 amino acid residues) was similar but not identical to that of PAP's extrapeptide. The cDNA of PAP II was expressed in E. coli. The growth of the transformants was strongly inhibited after induction of the gene. Furthermore, PAP II, which was produced in E. coli, inhibited protein synthesis in a rabbit reticulocyte translation system. Thus, recombinant PAP II would appear to be as functional as native PAP in inhibiting protein synthesis in both prokaryotes and eukaryotes.

Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation.

Eukaryotic translation initiation factor-4A (eIF-4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA-dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA-ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF-4F. To gain further understanding into the mechanism of action of eIF-4A in mRNA-ribosome binding, defective eIF-4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF-4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap-independent internal initiation mechanism. Addition of eIF-4A or eIF-4F relieved inhibition of translation, but eIF-4F was six times more effective than eIF-4A, whereas eIF-4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF-4A is defective in recycling through eIF-4F, thus explaining the dramatic inhibition of translation. Mutant eIF-4A proteins also inhibited eIF-4F-dependent, but not eIF-4A-dependent RNA helicase activity. Taken together these results suggest that eIF-4A functions primarily as a subunit of eIF-4F, and that singular eIF-4A is required to recycle through the complex during translation. Surprisingly, eIF-4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.

In vitro expression of the human cytomegalovirus DNA polymerase gene: effects of sequence alterations on enzyme activity

Genomic DNA of the Towne strain human cytomegalovirus polymerase (pol) gene (4.4-kb RsrII-NcoI segment of the EcoRI J fragment) was cloned into plasmids containing either the T3 or the T7 promoter for in vitro transcription-translation studies. The translation efficiency of unmodified pol cRNA was poor in this system and could not be improved by capping. However, the efficiency could be enhanced by replacing the leader sequence with a 40-bp AT-rich sequence derived from an alfalfa mosaic virus, R4. pol cRNA directed the synthesis of a 140-kDa polypeptide in a rabbit reticulocyte translation system. The in vitro-translated wild-type enzyme possessed significant polymerization activity which could be stimulated by salt as could that of the authentic enzyme purified from virus-infected cells. To study the critical domains of this enzyme, nine mutations were introduced into the pol gene around the conserved domains of eukaryotic polymerase by oligonucleotide-directed mutagenesis. Two constructs with mutations at amino acid residues 323 to 325 (M32QS) and 725 to 726 (M72II) remained active, with partial loss of enzyme activity, while the enzyme activities of other mutants with alterations at four domains located around amino acid residues 729 to 730 (M73HN), 804 to 807 (M80 and DE80), 910 to 913 (M91 and DE91), and 962 to 964 (M96 and DE96) were abolished. DNA template and triphosphate binding assays indicated that the mutation at 804 to 807 (conserved region III) lost the ability to bind DNA template, and four mutants, M73HN (within conserved region II), M80 (in region III), M91 (in region I), and M96 (around region V [962 to 964; amino acid sequence KKR]), failed to bind deoxyribonucleoside triphosphate. These data suggest that conserved region III is essential for DNA template binding, while residues between conserved region II and V (725 to 964) are involved in triphosphate binding.

Effects of ribosome-inactivating proteins on Escherichia coli and Agrobacterium tumefaciens translation systems.

The effects of 30 type 1 and of 2 (ricin and volkensin) type 2 ribosome-inactivating proteins (RIPs) on Escherichia coli and Agrobacterium tumefaciens cell-free translation systems were compared with the effects on a rabbit reticulocyte translation system. The depurinating activity of RIPs on E. coli ribosomes was also evaluated. Only six type 1 RIPs inhibited endogenous mRNA-directed translational activity of E. coli lysates, with submicromolar 50% inhibitory concentrations. Four RIPs had similar activities on poly(U)-directed phenylalanine polymerization by E. coli ribosomes, and three RIPs inhibited poly(U)-directed polyphenylalanine synthesis by A. tumefaciens ribosomes, with submicromolar 50% inhibitory concentrations.

Use of Nonreducing SDS-PAGE for Monitoring Renaturation of Recombinant Protein Synthesis Initiation Factor, eIF-4α

The purification of biologically active human protein synthesis initiation factor 4α, eIF-4α, overexpressed in Escherichia coli, is complicated by its localization in insoluble inclusion bodies, as well as its possession of four cysteines. Two of these cysteines have been reported to be reduced in the native molecule and two form a disulfide bond. A method is described, using nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis, for monitoring renaturation of the polypeptide during staged dialyses in decreasing urea concentrations. The production of biologically active eIF-4α only occurs when the polypeptide is totally reduced during solubilization of the inclusion bodies in 8 M urea. This requires a minimum dithiothreitol concentration of 50 mM. Conversely, reformation of the disulfide bonds only occurs when the staged dialyses are performed at lower concentrations of sulfhydryl reagents. Once renatured, as described, eIF-4α can be purified by affinity chromatography on m7GTP-Sepharose. Approximately 20 μg of biologically active eIF-4α per milliliter of bacterial culture can be obtained. The affinity-purified eIF-4α has activity equivalent to that reported for purified native human eIF-4α, as measured by its activity in a rabbit reticulocyte translation system. The method described is applicable to the purification of other cysteine-contaming polypeptides that accumulate to high levels in inclusion bodies.

Comparison of in vitro translation products of Sarcocystis gigantea and Sarcocystis tenella

RNA was purified from cystozoites of Sarcocystis gigantea and Sarcocystis tenella and used to in vitro translate polypeptides in a wheat germ and a rabbit reticulocyte translation system. The in vitro translated polypeptides were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The S. tenella mRNA translated at least two polypeptides (mol. wt about 80,000 and 21,500) in both translation systems that were not translated by the S. gigantea mRNA. To study co-translational and initial post-translational processing in Sarcocystis, the poly(A) + RNA preparations were in vitro translated in the rabbit reticulocyte translation system in the presence or absence of canine microsomal membranes. Based on electrophoresis, there appeared to be modification of at least some Sarcocystis polypeptides in the mol. wt range 17,000–30,000. In addition, the translation products were immunoprecipitated with a homologous and a heterologous antiserum. The immunoprecipitated polypeptides were compared by electrophoresis and the S. tenella translation products contained at least one unique antigenic polypeptide with a mol. wt of about 34,700 that was not processed by the microsomal membranes. These results suggest that there is at least one polypeptide that is a candidate for use as an antigen for the differentiation of S. gigantea and S. tenella infections in sheep.

Putative New Expression of Genes in Ixodid Tick Salivary Gland Development during Feeding

Poly(A+) mRNA-enriched fractions from salivary glands of partially fed Amblyomma americanum female ticks were translated in vitro with a rabbit reticulocyte translation system. Translated proteins were labeled with [35S]methionine, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and identified by autoradiography. Thirty major identifiable polypeptides with molecular weights ranging from 14 to 136 kDa were synthesized from mRNA isolated from salivary glands of ticks weighing less than 100 mg. Polypeptides that comigrated at the same molecular weight were translated by mRNA from ticks at a more advanced stage of feeding (more than 300 mg) as were 8 others with molecular weights of 31, 71, 91, 106, 113, 118, and 128 kDa. Results demonstrated that differential gene expression may be stimulated in the developing salivary glands as the tick feeds.