Vaporization of Inorganic Mercury by Cell-free Extracts of Drug Resistant Escherichia coli

The cDNAs encoding venom phospholipase A₂ (PLA₂) inhibitors (PLIs), named Protobothrops elegans (Pe)γPLI-A, PeγPLI-B, PeαPLI-A, and PeαPLI-B, were cloned from the P. elegans liver cDNA library. They were further divided into several constituents due to nucleotide substitutions in their open reading frames. For PeαPLI-A, two constituents, PeαPLI-A_a and PeαPLI-A_b, were identified due to three nonsynonymous substitutions in exon 3. Far-western blot and mass-spectrometry analysis of the P. elegans serum proteins showed the presence of γPLIs, and αPLIs, which can bind venom PLA₂s. In αPLIs from Protobothrops sera, A or B subtype-specific amino acid substitutions are concentrated only in exon 3. A comparison of γPLIs showed that γPLI-As are conserved and γPLI-Bs diversified. Mathematical analysis of the nucleotide sequences of Protobothrops γPLI-B cDNAs revealed that the particular loops in the three-finger motifs diversified by accelerated evolution. Such evolutionary features should have made serum PLIs acquire their respective inhibitory activities to adapt to venom PLA₂ isozymes.     

In Vitro Translation of Cardiovirus Ribonucleic Acid by Mammalian Cell-free Extracts

Cell-free extracts prepared from Ehrlich ascites and mouse L cells synthesize viral proteins in response to encephalomyocarditis virus, mouse Elberfeld virus, and mengovirus ribonucleic acid. Although HeLa cell extracts are inactive, their ribosomes are functional in the presence of heterologous supernatant fractions. Synthesis depends upon the addition of adenosine triphosphate, guanosine triphosphate, an energy-generating system, and 4 mm Mg(2+). Initiation is completed during the first 10 to 20 min of incubation, but chain elongation continues for 1 hr or more. The products are of higher molecular weight than virion structural proteins and resemble polypeptides formed in virus-infected cells during a short pulse. Tryptic peptides of virion proteins and in vitro products are similar for all three cardioviruses.     

Tumour Induction by Cell-free Extracts derived from Mice with Graft versus Host Disease

A SUBSTANTIAL proportion of inbred mice, in which a protracted form of the graft versus host reaction (GVHR) is induced, eventually develop lymphoreticular tumours¹. This experimental mouse model can be used to study the role of immune reactions in cancer induction. One possibility is that the immune disturbance activates a latent oncogenic virus. That such activation can occur has been demonstrated in C57BL mice². In this strain, with its low spontaneous leukaemic incidence, thymic lymphomas are induced by an irradiation-activated leukaemogenic virus. In man, there is suggestive evidence that Burkitt's lymphoma may be caused by a combination of virus infection and immunological stimulation³.     

Isolation and Cell-free Translation of Total Messenger RNA from Germinating Castor Bean Endosperm

Polyadenylated RNA was isolated from the total RNA fraction extracted from the endosperm tissue of 3-day-old castor bean seedlings by affinity chromatography on oligo(dT)-cellulose. This polyadenylated RNA was efficiently translated into protein when added to a messenger RNA-dependent cell-free system derived from rabbit reticulocytes. Characterization of the translational products by electrophoresis followed by autoradiography established that numerous discrete polypeptides were formed with molecular weights ranging from 10,000 to over 100,000. Immunoprecipitation in the presence of antiserum raised in rabbits against the total glyoxysomal matrix proteins showed that these proteins accounted for 15 to 20% of the total translational products.     

Potyviral VPg enhances viral RNA Translation and inhibits reporter mRNA translation in planta

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.     

Translation of Globin Messenger RNA in a Heterologous Cell-free System

MESSENGER-SPECIFIC initiation factors, capable of discriminating between classes of messenger RNAs (mRNAs) or different cistrons in viral RNA, have been implicated in the regulation of protein synthesis in bacteria^1–5. Comparable but less detailed observations have also been made in eukaryotic systems^6–10. For example, RNA extracted from a mammalian virus (encephalomyocarditis virus, EMC) cannot be translated in a reticulocyte cell-free system unless the system is fortified with an extract from responsive cells—in this case, Krebs II ascites cells⁶. Such results imply the existence of tissue-specific factors and lead to questions whether this incompatibility is reciprocated by an inability of the Krebs II ascites cell system to respond to the mRNA for globin.     

Variation of Cell-free Translation Profiles Among Pathogenic Strains of Soybean Mosaic Virus

Cell-free translation products from isolates representing soybean mosaic virus (SMV) strains G1 to G7 and G7a, along with several other SMV isolates, were analyzed. SMV RNAs were translated in both rabbit reticulocyte lysates and wheat germ extracts, yielding approximately 20 translation products for each strain from each translation system. Comparison of translation profiles by the presence or absence of proteins allowed for the formation of distinctive groups from each cell-free translation system. Groupings formed by analysis of products from rabbit reticulocyte lysates correlated with pathogenicity; groupings formed by analysis of products from wheat germ extracts had no apparent biological significance.     

Translation of Vesicular Stomatitis Messenger RNA by Extracts from Mammalian and Plant Cells

RNA was isolated from polyribosomes of vesicular stomatitis virus (VSV)-infected cells and tested for its ability to direct protein synthesis in extracts of animal and plant cells. In cell-free, non-preincubated extracts of rabbit reticulocytes, the 28S VSV RNA stimulated synthesis of a protein the size of the vesicular stomatitis virus L protein whereas the 13 to 15S RNA directed synthesis of the VSV M, N, NS, and possibly G proteins. In wheat germ extracts, 13 to 15S RNA also directed synthesis of the N, NS, M, and possibly G proteins. Analysis of extracts labeled with formyl [(35)S]methionine showed that the 28S RNA directed the initiation of synthesis of one protein, whereas the 13 to 15S RNA directed initiation of at least four proteins. It is concluded that the 28S RNA encodes only the L protein, whereas the 13 to 15S RNA is a mixture of species, presumably monocistronic, which code for the four other known vesicular stomatitis virus proteins.     

Terpene Biosynthesis in Cell-free Extracts and Excised Shoots from Wedgwood Iris

Excised shoots and cell-free extracts prepared from Wedgwood iris (Iris hollandica Hoog. “Wedgwood”) shoots metabolized ¹⁴C-labeled mevalonic acid (MVA). By using cell-free extracts, the ¹⁴C from MVA-1-¹⁴C was recovered as ¹⁴CO₂, while that from MVA-2-¹⁴C was recovered as neutral terpenes, acid-hydrolyzable terpenes, or ¹⁴CO₂. Also, under optimal incubation conditions, 12.8 nanomoles R-MVA-2-¹⁴C was incorporated into neutral terpenes per milligram fresh weight per hour. In contrast, excised shoots incorporated only 0.58 nanomoles R-MVA-2-¹⁴C per mg fresh weight per hour. Labeled products identified from the cell-free system were squalene, farnesol, geranylgeraniol, and compounds that are converted to farnesol and geranylgeraniol after alkaline hydrolysis. Squalene and a 4,4-dimethylsterol were identified as products from excised shoots but not the terpene alcohols or the alkaline-hydrolyzable compounds.     

Biosynthesis of Indoleacetic Acid from Tryptophan-C in Cell-free Extracts of Pea Shoot Tips

A 2-step, 1-dimensional thin-layer chromatographic procedure for isolating indoleacetic acid (IAA) was developed and utilized in investigations of the biosynthesis of IAA from tryptophan-¹⁴C in cell-free extracts of pea (Pisum sativum L.) shoot tips. Identification of a ¹⁴C-product as IAA was by (a) co-chromatography of authentic IAA and ¹⁴C-product on thin-layer chromatography, and (b) gas-liquid and thin-layer chromatography of authentic and presumptive IAA methyl esters. Dialysis of enzyme extracts and addition of α-ketoglutaric acid and pyridoxal phosphate to reaction mixtures resulted in approximately 2- to 3-fold increases in net yields of IAA over yields in non-dialyzed reaction mixtures which did not contain additives essential to a transaminase reaction of tryptophan. Addition of thiamine pyrophosphate to reaction mixtures further enhanced net biosynthesis of IAA. It is concluded that the formation of indolepyruvic acid and its subsequent decarboxylation probably are sequential reactions in the major pathway of IAA biosynthesis from tryptophan in cell-free extracts of Pisum shoot tips. Comparison of maximum net IAA biosynthesis in extracts of shoot tips of etiolated and light-grown dwarf and tall pea seedlings revealed an order, on a unit protein N basis, of: light-grown tall > light-grown dwarf > etiolated tall etiolated dwarf. It is concluded that the different rates of stem elongation among etiolated and light-grown dwarf and tall pea seedlings are correlated, in general, with differences in net IAA biosynthesis and sensitivity of the tissues to IAA.     

Cell-free Cytoplasmic Polyadenylation of Oogenic RNA

The products of cell-free ATP incorporation mediated by cytoplasmic fractions prepared from unfertilized sea urchin eggs, anucleate egg halves, nucleate egg halves, emetine-treated fertilized eggs, and four-cell embryos have been characterized to determine to what extent the polymers synthesized are poly(A) and to assess the size distribution of the primers adenylated. As judged by alkaline lability, ribonuclease resistance, and retention on poly(U)-impregnated filters, greater than 92% of the label recovered after RNA extraction is present in poly(A). LiCl fractionation indicates that little, if any, free poly(A) is synthesized or cleaved from RNA primers during the reaction, and that 4S RNA is not an effective initiator. In excess of 85% of the poly(A) is associated with RNA having S-values greater than or equal to 18S. Sedimentation profiles of RNA adenylated in the unfertilized egg and anucleate egg half reactions are identical. Suppression of in vivo protein synthesis by emetine alters the profile of RNA subsequently adenylated in vitro. It is proposed that the apparent constraints on the utilization of cytoplasmic RNA or ribonucleoprotein primers of oogenic origin may be effected by RNA-associated proteins capable of regulating the selection and/or extent of their polyadenylation during early embryogenesis.     

Biosynthesis of Desthiobiotin in Cell-free Extracts of Escherichia coli

Cell-free extracts of Escherichia coli were active in catalyzing the synthesis of a biotin vitamer from 7,8-diaminopelargonic acid. The vitamer was identified as desthiobiotin on the basis of its chromatographic and electrophoretic characteristics and its biotin activities for a variety of microorganisms. The reaction was stimulated five-fold by bicarbonate, suggesting that an "active CO(2)" was incorporated into the carbonyl carbon of desthiobiotin. The enzyme was demonstrable in a wild-type (K-12) and in all biotin mutants of E. coli that were tested, with the exception of a strain which was able to grow on desthiobiotin but not on diaminopelargonic acid. Furthermore, the enzyme was repressible by biotin in all of the strains tested. These results are consistent with the hypothesis that the biosynthesis of desthiobiotin from 7,8-diaminopelargonic acid is an obligatory step in the biosynthetic pathway of biotin in E. coli.     

Cell-free translation products of basement membrane RNA from the EHS tumor

The biosynthetic products of the Engelbreth-Holm-Swarm (EHS) tumor and the cell-free translation products of EHS tumor cell RNA were characterized. Six distinct gene products (three laminin polypeptides, entactin/nidogen, and two collagen IV chains) comprising the basement membrane matrix were identified by a combination of proteolytic digestion and immunologic techniques. Analysis of the cell-free translation products using EHS tumor RNA precipitated by anti-laminin serum confirms earlier evidence that there are at least two B chains encoded by different genes. The anti-laminin serum also recognized entactin/nidogen, which was further identified by specific immunoprecipitation with anti-entactin serum. Radiolabeled laminin A chains, synthesized by the EHS tumor in organ culture, were also identified by the anti-laminin serum but were not detected among the cell-free translation products of EHS tumor RNA. Pulse-chase studies of EHS tumor in organ culture as well as in vitro translation of EHS tumor RNA suggest that the precursor forms of alpha 1(IV) and alpha 2(IV) collagen chains are nearly identical in size, with apparent molecular weights of 170,000. The mRNAs encoding these two polypeptides migrate differently on sucrose gradients. It is likely that glycosylation and hydroxylation of collagen IV account for the major differences in molecular weight of mature alpha 1(IV) and alpha 2(IV) chains in the EHS tumor matrix.     

Cell-free translation of murine coronavirus RNA.

The coding assignments of the intracellular murine hepatitis virus-specific subgenomic RNA species and murine hepatitis virion RNA have been investigated by cell-free translation. The six murine hepatitis virus-specific subgenomic RNAs were partially purified by agarose gel electrophoresis and translated in an mRNA-dependent rabbit reticulocyte lysate, and the cell-free translation products were characterized by gel electrophoresis, immunoprecipitation, and tryptic peptide mapping. These studies have shown that RNA 7 codes for the nucleocapsid protein, RNA 6 codes for the E1 protein, RNA 3 codes for the E2 protein, and RNA 2 codes for a 35,000-dalton nonstructural protein. Genomic RNA directs the cell-free synthesis of three structurally related polypeptides of greater than 200,000 in molecular weight.     

Cell-free translation of avian erythroblastosis virus RNA

Avian erythroblastosis virus (AEV) RNA rescued from nonproducer cells by superinfection with a helper virus is translated into three polypeptides in the messenger-dependent rabbit reticulocyte lysate. A 75,000 molecular weight polypeptide (P75AEV) is synthesized from 28S RNA and is encoded by the 5' section of the AEV RNA, including gag-related and AEV-specific sequences. The P75AEV synthesized in infected cells and the P75AEV synthesized in the cell-free system are electrophoretically identical. A 44,000 molecular weight polypeptide (P44AEV) is synthesized from 20-24S RNA, apparently from the 3' section of the AEV-specific RNA sequence. A minor 37,000 molecular weight polypeptide (P37AEV) is synthesized from 20S AEV RNA. A comparison is drawn between the cell-free products of MC29 and AEV RNAs.     

Cell-free translation of messenger RNA from oocytes of Xenopus laevis

The poly(A)⁺ RNA which accumulates during oogenesis in the amphibian Xenopus laevis is shown to be functional mRNA; the RNA was active in the mRNA-dependent “shift assay” for initiation sites in the rabbit reticulocyte lysate, and was an efficient template for protein synthesis in the wheat-germ cell-free system. Analysis of the in vitro protein products showed no differences between the coding properties of poly(A)⁺ RNA extracted from oocytes at all stages of development from previtellogenesis to maturity. In previtellogenic oocytes, the in vitro products of polysomal and of mRNP-associated poly(A)⁺ RNA were also identical. Neither was there any evidence for changes in the coding properties of the poly(A)⁺ mRNA of the oocyte. However, the patterns of oocyte in vivo protein synthesis changed markedly during early vitellogenesis. We conclude that the mRNP-associated poly(A)⁺ RNA present in mature oocytes constitutes the stored maternal mRNA, and that during oogenesis the coding composition of the poly(A)⁺ mRNA synthesised does not change markedly, while some form of translational control operates to direct the changing pattern of protein synthesis.     

Photoregulation of Nicotinamide Adenine Dinucleotide Kinase Activity in Cell-free Extracts

This article gives evidence that NAD kinase activity is controlled by the action of phytochrome. The NADP level rapidly increased in the cotyledons of seedlings of Pharbitis nil strain Violet (a short day plant), when the inductive dark for flowering was interrupted with a 5-minute illumination of red light. Illumination with far red light immediately after illumination with red light counteracted partly the effect of the latter.