Molecular and biochemical aspects of nematode collagens.

Collagens are major structural proteins of nematode cuticles and basement membranes (basal laminae). The collagen proteins that form these structures differ in their biochemical and physical properties and are encoded by distinct gene families. Nematode basement membrane collagens are large proteins that show strong homology to basement membrane collagens of vertebrates. There appear to be 2 nonidentical basement membrane collagen genes in nematodes. Cuticle collagens are about one-sixth the size of basement membrane collagens and are encoded by a large family of 20-150 nonidentical genes. Cuticle collagens can be subdivided into 4 families based upon certain structural features in the proteins. The mature, extracellular forms of both types of collagen proteins are extensively cross-linked by disulfide bonds and are largely insoluble in the absence of a thiol-reducing agent. Cuticle collagens also are cross-linked by nonreducible covalent bonds that involve tyrosine residues. The experimental studies that have led to our current understanding of the structures of basement membrane and cuticle collagens are reviewed. Some previous questions about the physical properties of these proteins are reexamined in light of the primary sequence information now available for the proteins.     

In vitro translation with adenovirus polyribosomes.

Polyribosomes isolated from adenovirus type 2 (Ad2)-infected HeLa cells late in productive infection can be used for translation in cell-free systems. At least eight viral polypeptides are synthesized, including the precursors to virion polypeptides VI and VII. Separation of polyribosomes by zonal rate centrifugation followed by translation in a cell-free system reveals a correlation between the sizes of the polyribosomes and the polypeptides synthesized. The cell-free extracts incorporate amino acid linearly for only 10 min and show little or no capacity to reinitiate protein synthesis. The elongation efficiency measured as the number of amino acids incorporated per ribosome in 20 min is low, ranging from 10 to 100. The maximum chain elongation rate is estimated to be 10 to 20 amino acids per min. The limited elongation has been used to assess the relative concentration of mRNA's engaged in translation.     

In vitro translation of mushroom tyrosinase

Mushroom tyrosinase was purified and antibodies prepared against the holo enzyme and a protein of 26,000 daltons. Both antibodies recognized the large subunit of the enzyme but only one recognized the 26,000 dalton protein. Poly A+ mRNA was isolated from mushrooms, translated in vitro, and a 41,000 dalton protein immunoprecipitated from the translation mix with either antibody. This 41,000 dalton protein presumably corresponds to the large subunit of the holoenzyme. Antibodies against the holoenzyme also immunoprecipitated another translation product with a molecular weight of 15,000 daltons corresponding to the small subunit of the holoenzyme. These results suggest that each subunit may be coded for by different genes and undergo posttranslational processing.     

In vitro translation of Harvey murine sarcoma virus RNA.

The viral RNA of the Harvey strain of murine sarcoma virus (Ha-SV), which does not encode for any known viral structural polypeptides, has been translated in a nuclease-digested, cell-free system. The major protein product of the in vitro translation reaction has a molecular weight of 21,000 and is initiated faithfully with [35S]formylmethionine from formyl-[35S]methionyl-tRNAFMET. This polypeptide is clearly distinct from the RNA of the Moloney strain of type C helper virus used to pseudotype the Ha-SV. The intensity of the 21,000-dalton polypeptide on gels correlates well to the concentration of Ha-SV RNA in different viral RNA preparations. These experiments indicate that a polypeptide marker for Ha-SV is now available for the first time. The possibility that this protein is the product of the rat portion of the Ha-SV genome is discussed.     

In vitro translation of the three bacteriophage phi 6 RNAs.

In vitro translation of the three single-stranded RNAs transcribed in vitro by bacteriophage phi 6 RNA polymerase revealed that the large RNA codes for phage proteins P1, P2, P4, and P7, the medium RNA codes for P3, P6, and P10, and the smaller RNA for P5, P8, and P9.     

In vitro translation of intestinal sucrase-isomaltase and glucoamylase

It is has been proposed that both sucrase-isomaltase and glucoamylase are initially synthesized as large single-chain precursors which are then processed to heterodimers. We have tested this hypothesis by in vitro translation of their mRNAs. The primary translation product of sucrase-isomaltase mRNA was a single polypeptide of Mr = 190,000. Similar experiments using antiserum against glucoamylase revealed a single polypeptide of Mr = 145,000. These results are consistent with the single chain precursor hypothesis for sucrase-isomaltase. However, the glucoamylase product (145 kDa) is too small to be processed to a heterodimer of Mr = 230,000. Moreover, the mature subunits (Mr = 135,000 and 125,000) probably are derived from the 145 kDa precursor by proteolytic trimming and must include and share most of the precursor protein.     

In vitro maintenance of nematode parasites assayed by acetylcholinesterase and allergen secretion.

A comparison was made of the ability of Nippostrongylus brasiliensis and Necator americanus to synthesize and secrete acetylcholinesterase when they were maintained in different in vitro culture media. The amount of allergen released by N. brasiliensis was also studied. The adult and fourth stage larval stages (but not the infective larvae) of Necator and adult N. brasiliensis secreted from their anterior glands up to 40 times as much acetylcholinesterase as they contained at the outset of culture. In contrast, allergen, which is less easy to quantitate, was secreted into the same media at about one-third the rate of secretion of acetylcholinesterase. Acetylcholinesterase was synthesized and released by both worms in media containing protein and the enzyme did not lose activity when kept for several days at 37 C. The secretion of this enzyme by nematodes kept in culture provides a simple, sensitive, rapid, and quantitative assay for measuring the ability of these nematodes to synthesize and secrete antigens in culture.     

In vitro translation of the intermediate filament proteins desmin and vimentin.

Polyadenylated ribonucleic acid (RNA) was isolated from chicken skeletal and smooth muscle and translated in a cell-free rabbit reticulocyte system. Both types of muscle tissue contain messenger RNAs that code for the intermediate filament proteins desmin and vimentin, and the relative concentrations of the two translation products reflect the prevalence of the two proteins in vivo. Desmin synthesis represents a greater proportion of the total protein synthesis from smooth muscle RNA than from skeletal muscle RNA, whereas the converse is true of vimentin synthesis. Fractionation of the RNA on formamide-containing sucrose gradients before translation indicates that the desmin messenger RNA is larger than the vimentin messenger RNA and contains an extensive noncoding segment. The desmin and vimentin messages code predominantly for the non-phosphorylated forms of desmin and vimentin. However, the ratio of phosphorylated to unphosphorylated forms of the proteins could be increased by adding cyclic adenosine monophosphate-dependent kinase activity to the translation mixtures. These results suggest that desmin and vimentin are each synthesized from a single messenger RNA species and that posttranslational phosphorylation generates the additional isoelectric variants of each which are observed in vivo.     

In vitro assessment of plant lectins with anti-pinwood nematode activity

Two lectin proteins were purified from the corms of Pinellia ternata and Lycoris radiata. Both P. ternata agglutinin (PTA) protein and L. radiata agglutinin (LRA) protein formed polymers and coagulated both rabbit red blood cells and yeast cells. The two proteins were each diluted to different concentration and then mixed with pinewood nematodes, and nematode survival was measured. Results showed that the two lectin proteins showed significant levels of resistance against nematodes and the nematode population was significantly reduced, compared to PBS buffer without protein control group. The mean number of nematodes of two lectin proteins group was significantly lower than that of control group constantly throughout the assay period with differences being very significant at P < 0.01 after 24 h. After 96 h, when 500 μg/ml proteins were used, nematode number significantly declined to an average of 26 (approximately 43% of the controls) and 32.2 (approximately 53.3% of the controls) nematodes at LRA and PTA protein, respectively, compared to the control group. Results also indicated that higher concentrations of protein were more toxic to the pinewood nematode. Even when the concentration was as low as 30 μg/ml, the toxic proteins retained their anti-nematode activity. Furthermore, pinewood nematode was exposed to the proteins for longer, more pinewood nematodes were killed. Our results indicated the two lectin proteins both apparently have a toxic effect on the pinewood nematode that affects its survival in vitro.     

In vitro translation of rat lung surfactant apoprotein mRNA

Rat pulmonary surfactant contains apoproteins of molecular weights 38,000, 32,000, 26,000 and 10,000-12,000. The structural and metabolic interrelationships of these proteins are not clear as yet. In order to investigate if they arise from a single or multiple precursor protein (s), we isolated total poly(A)RNA from rat lungs, performed its translation in vitro in the presence of [35s]-methionine and reticulocyte lysate, immunoprecipitated the translation products with anti-rat surfactant antibody, and analyzed them by SDS-polyacrylamide gel electrophoresis and autoradiography. A single translation product of molecular weight 35,000 was detected. Since the antibody used in the immunoprecipitation recognizes the 38,000, 32,000 and 26,000 dalton proteins, it is concluded that at least these three proteins arise from the 35,000 dalton precursor by post-translational modifications.     

In vitro translation studies of the cytoplasmic nonpolysomal particles containing messenger RNA.

We analyzed the translational capacity of different kinds of free cytoplasmic messenger ribonucleoprotein complexes (free mRNP) in a Hela cell cell free system. Native free mRNP are not translated although free mRNP washed with 0.5 M KC1 can direct polypeptide synthesis. Furthermore, the 0.5 M KC1 wash possesses a factor which inhibits the translation of 0.5 M KC1 washed free mRNP as well as globin mRNA naked mRNA from plasmocytoma, or Hela cells. We also demonstrated that native free mRNP are able to form a complex with ribosomal subunits in the presence of initiation factors. This indicates that inhibition of translation by the 0.5 M KC1 wash occurs either at some point after initiation complex formation or at the elongation step.     

In situ assembly of cuticular wax

Summary Cytochemical reactions within the primary cuticle (cutinised layer) indicate that the lamellae are formed from polar lipids. The electron microscope shows that the lamellae are involved in wax formation and it is suggested that the polar lipids provide in situ precursors for the synthesis of cuticular wax.     

Isolation and translation in vitro of poly(A)+RNA from the free-living nematode Panagrellus silusiae.

Polysomal RNA was isolated from the free-living nematode Panagrellus silusiae. Passage of this RNA through a cellulose column resulted in the fractionation of the input RNA into poly(A)-RNA (ca. 97.5% of the total) and poly(A)+ RNA (ca. 2.5% of the total). RNase digestion, followed by polyacrylamide gel electrophoresis, revealed that the poly(A)+ RNA contained poly(A) tracts that ranged from 75 to 104 nucleotides in length with a mean value of about 90 residues. There was no evidence of poly(A) sequences in the poly(A)- RNA fraction. Poly(A)+ RNA gave a 25- to 50-fold stimulation (over background) of amino acid incorporation in the wheat germ cell-free protein-synthesizing system. At least 26 proteins were evident after electrophoresis in cylindrical sodium dodecyl sulfate-polyacrylamide gels. Poly(A)-RNA was capable of stimulating protein synthesis in vitro with about five discrete proteins being produced. In summary, the properties of mRNA from a simple organism such as P. silusiae are very similar to those of more complex eukaryotes.     

In vitro translation and processing of rat kidney gamma-glutamyl transpeptidase

Rat kidney gamma-glutamyl transpeptidase is composed of two nonidentical glycosylated subunits. The enzyme is localized on the lumenal surface of the brush-border membranes of proximal tubule epithelial cells; it is attached to the membranes via an NH2-terminal segment of the larger of the two subunits. Tissue-labeling experiments followed by immunoprecipitation with antibodies directed against the enzyme and its two subunits demonstrate that a glycosylated single chain precursor (Mr = 78,000), containing the elements of both the subunits, is initially synthesized. Pulse-chase studies in the presence of pactamycin, and inhibitor of protein synthesis initiation, indicate that the larger of the two subunits is located at the NH2 terminus of the Mr = 78,000 precursor. The initial events in the biosynthesis and processing of gamma-glutamyl transpeptidase were investigated by in vitro translation of rat kidney mRNA. Such translation results in the synthesis of a Mr = 63,000 unglycosylated polypeptide which has been shown immunologically to contain the domains for both subunits. The Mr = 63,000 species is processed to a Mr = 78,000 core-glycosylated polypeptide when translation of mRNA is carried out in the presence of dog pancreas microsomes. This processing does not appear to be associated with cleavage of an NH2-terminal leader sequence. The Mr = 78,000 polypeptide is integrated into the microsomal membranes with an orientation that is analogous to that found on the brush-border membranes. Glycosylation and membrane integration of transpeptidase are cotranslational events. Upon longer incubation, the Mr = 78,000 species sequestered within the microsomal vesicles is cleaved to species corresponding in size to the two subunits of the kidney enzyme.     

In vitro translation products specified by the transforming region of adenovirus type 2.

Region 1 DNA sequences (map positions 0 to 11% on the linear adenovirus 2 genome) are expressed both early and late in lytic infection and are required for transformation by the virus. During productive infection six distinct cytoplasmic RNAs are synthesized from this region. These RNAs comprise two families, each consisting of three size classes that share 3' sequences. Region 1 RNA's were purified by hybridization selection, using restriction fragments bound to nitrocellulose membranes, and by size fractionation. The isolated RNAs were then translated in cell-free systems derived from wheat germ and rabbit reticulocytes. The family of RNAs specified by 0 to 4.4 sequences includes two RNAs, which are 12S and 13S in size. These RNAs were partially separated by molecular weight and translated. The 13S RNA produced 53,000-dalton (53K) and 41K peptides, and the 12S RNA synthesized 47K and 35K products. The family of RNAs mapping from 4.4 to 11.0 encodes three separate polypeptides, each of which can be assigned to a specific RNA. A 12K product that comigrates with structural polypeptide IX is synthesized from the 9S RNA as previously reported (U. Pettersson and M. B. Mathews, Cell 12:741-750, 1977). The 13S RNA encodes a 15K polypeptide that corresponds to a 15K polypeptide in infected cell extracts. The 22s RNA encodes a 52K protein distinct from the 0 to 4.4 polypeptides.     

In vitro translation of archaeal natural mRNAs at high temperature

Abstract Efficient in vitro translation of archaeal natural mRNAs at high (75°C) temperature has been achieved by employing either crude cell lysates or purified ribosomes and soluble proteins of the extreme thermophile Sulfolobus solfataricus . The features of the system are described.