Cell-free synthesis of phytochrome apoprotein

A single polypeptide is immunospecifically precipitated by monospecific antiphytochrome from the total translation products of both wheat-germ and rabbit-reticulocyte cell-free protein synthesizing systems programmed with oat (Avena sativa L.) poly(A) RNA. The mobility of this polypeptide is slightly lower on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis than that of immunoaffinity-purified, 118 kdalton phytochrome and corresponds to an apparent molecular weight of 124 kdalton. Evidence against the possibility that this mobility difference results from intracellular processing of the 124-kdalton protein is provided by extraction of freeze-dried tissue directly into boiling SDS-containing buffer. This procedure yields a phytochrome species with a mobility on SDS polyacrylamide gel electrophoresis indistinguishable from that of the in-vitro translation product. Together the data indicate that the phytochrome polypeptide is synthesized in its mature form in the cell but is subject to modification to a form with lower apparent molecular weight during immunopurification.Abbreviations IgG immunoglobulin G - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Ribonucleic acid synthesis in chloroplasts

Chloroplasts isolated from young spinach leaves incorporate [(3)H]uridine into RNA. This incorporation shows an absolute requirement for light and does not occur in lysed chloroplasts. Fractionation by polyacrylamide-gel electrophoresis of the RNA synthesized in vitro reveals a major discrete product of molecular weight 2.7x10(6) and two minor products of molecular weight 1.2x10(6) and 0.47x10(6). These discrete products are super-imposed on a background of polydisperse RNA. The incorporation of (32)P(i) into chloroplast rRNA species (mol.wt. 1.05x10(6) and 0.56x10(6)) in excised spinach leaves proceeds after a distinct lag period compared with the incorporation into cytoplasmic rRNA species (mol.wt. 1.34x10(6) and 0.7x10(6)). Incorporation of (32)P(i) into chloroplast RNA species of molecular weight 2.7x10(6), 1.2x10(6), 0.65x10(6) and 0.47x10(6) proceeds without such a time-lag. The kinetics of labelling of the individual RNA components is consistent with the rapidly labelled RNA species of molecular weight 1.2x10(6) and 0.65x10(6) being precursors to the more slowly labelled rRNA species of molecular weight 1.05x10(6) and 0.56x10(6) respectively.     

Structure and proteolysis of the growth hormone receptor on rat hepatocytes

125I-Labeled human growth hormone is isolated in high molecular weight (Mr) (300,000, 220,000, and 130,000) and low molecular weight complexes on rat hepatocytes after affinity labeling. The time-dependent formation of low molecular weight complexes occurred at the expense of the higher molecular weight species and was inhibited by low temperature or inhibitors of serine proteinases. Exposure to reducing conditions induced loss of Mr 300,000 and 220,000 species and augmented the amount of Mr 130,000 complexes. The molecular weight of growth hormone (22,000) suggests that binding had occurred with species of Mr 280,000, 200,000, and 100,000. Two-dimensional gel electrophoresis demonstrated that the 100,000-dalton receptor subunit is contained in both the 280,000- and 200,000-dalton species. Reduction of interchain disulfide bonds in the growth hormone receptor did not alter its elution from gel filtration columns, but intact, high molecular weight receptor constituents were separated from lower molecular weight degradation products. Digestion of affinity-labeled growth hormone-receptor complexes with neuraminidase increased the mobility of receptor constituents on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These observations show that the growth hormone receptor is degraded by hepatic serine proteinases to low molecular weight degradation products which can be separated from intact receptor by gel filtration. Intact hormone-receptor complexes are aggregates of 100,000-dalton sialoglycoprotein subunits held together by interchain disulfide bonds and by noncovalent forces.     

Comparison of mammalian mitochondrial ribosomal ribonucleic acid from different species

Mitochondrial ribosomal RNA species from mouse L cells, rat liver, rat hepatoma, hamster BHK-21 cells and human KB cells were examined by electrophoresis on polyacrylamide-agarose gels and sedimentation in sucrose density gradients. The S(E) (electrophoretic mobility) and S values of mitochondrial rRNA of all species were highly dependent on temperature and ionic strength of the medium; the S(E) values increased and the S values decreased with an increase in temperature at a low ionic strength. At an ionic strength of 0.3 at 23-25 degrees C or an ionic strength of 0.01 at 3-4 degrees C the S and S(E) values were almost the same being about 16.2-18.0 and 12.3-13.6 for human and mouse mitochondrial rRNA. The molecular weights under these conditions were calculated to be 3.8x10(5)-4.3x10(5) and 5.9x10(5)-6.8x10(5), depending on the technique used. At 25 degrees C in buffers of low ionic strength mouse mitochondrial rRNA species had a lower electrophoretic mobility than those of human and hamster. Under these conditions the smaller mitochondrial rRNA species of hamster had a lower electrophoretic mobility than that of human but the larger component had an identical mobility. Mouse and rat mitochondrial rRNA species had identical electrophoretic mobilities. Complex differences between human and mouse mitochondrial rRNA species were observed on sedimentation in sucrose density gradients under various conditions of temperature and ionic strength. Mouse L-cell mitochondrial rRNA was eluted after cytoplasmic rRNA on a column of methylated albumin-kieselguhr.     

Different Forms of Adrenal Phenylethanolamine N-Methyltransferase: Species-Specific Posttranslational Modification

Phenylethanolamine N-methyltransferase was purified from rat and cow adrenal glands. The enzymes from the two species have the same molecular weight of 31,000, but differ in electrophoretic mobility. During polyacrylamide gel electrophoresis, the rat form migrates faster than the bovine form. Antibodies to bovine enzyme precipitated equally well the rat and cow form of the enzyme, but antibodies against rat enzyme precipitated poorly the bovine form. In contrast, both antibodies recognized a similar protein in the in vitro translation products of poly(A+)mRNA isolated from cow adrenal glands. The results suggest that the primary protein structure of rat and bovine enzyme is similar and that differences in electrophoretic mobility are due to posttranslational modification of the enzyme molecule.     

Chloroplast ribosomal ribonucleic acid synthesis in cultured spinach leaf tissue

Chloroplast rRNA synthesis was studied in spinach leaf tissue cultured under sterile conditions which eliminate bacterial rRNA synthesis. The synthesis was inhibited by darkness, but concomitant cytoplasmic rRNA synthesis was unaffected. A complex pattern of labelled rRNA precursors was found in extracts from cultured leaf tissue by using polyacrylamide-gel electrophoresis. However, differences between the precursor profiles of leaf tissue cultured in the light and in the dark could not be correlated with chloroplast rRNA synthesis since large amounts of high-molecular-weight precursors of cytoplasmic rRNA dominated the pattern in both cases. A double-isotope-labelling technique was used, which enabled light-stimulated rRNA synthesis to be studied in whole leaf tissue. Two rapidly labelled RNA species of molecular weights 1.15x10(6) and 0.65x10(6) were detected, which were thought to have possible precursor significance in the synthesis of mature chloroplast rRNA of molecular weights 1.04x10(6) and 0.56x10(6) respectively. Cycloheximide treatment resulted in the accumulation of RNA of molecular weight 1.8x10(6), whose function is unknown.     

Ribosomal RNA Synthesis in the Eastern North-American Newt, Notophthalmus viridescens

Ribosomal RNA (rRNA) synthesis, the initiation of which is an early major event during the transformation of iris into lens in the newt, was characterized in the TVI cell-line derived from the eastern North-American newt Notophthalmus viridescens. Employing the technique of polyacrylamide gel electrophoresis, molecular-weight measurements were made on newt rRNAs using Xenopus laevis and E. coli rRNAs as standards. The molecular weights of N. viridescens 28S and 18S rRNA were found to be 1.4 × 10⁶ and 0.7 × 10⁶ respectively. The precursor to these RNAs had a molecular weight of 3.1 × 10⁶. Three probable intermediates in the processing of precursor to mature rRNA were also identified. On the basis of the molecular weights of all species of RNA identified, a processing pathway, similar to that of Xenopus , has been suggested.
Some unusual features in the kinetics of precursor rRNA labelling and processing suggest the possibility that newt-cell rRNA synthesis may be controlled by the availability of essential amino acids in a manner similar to that observed in mammalian cells. A possible relationship between the availability of essential amino acids, the initiation of rRNA synthesis in the newt iris, and the control of lens regeneration is discussed.     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Separation of Bacterial Ribosomal Ribonucleic Acid from Its Macromolecular Precursors by Polyacrylamide Gel Electrophoresis

Electrophoresis on polyacrylamide gels was found to be a powerful technique for separating the mature from the precursor forms of bacterial ribosomal nucleic acid (rRNA). The separation of the 16S rRNA from its precursor was, for all practical purposes, complete; that of the 23S rRNA from its precursor was detectable but incomplete. When mature and precursor rRNA preparations were heated to randomize secondary structure, etc., and then cooled, it was found that electrophoretic mobility differences between mature forms of rRNA and their precursors persisted. This, in conjunction with the rather large electrophoretic mobility differences between mature and precursor forms, can be taken as strong evidence for a molecular weight difference between mature rRNA and its precursor forms of RNA. With the 16S rRNA, this difference could be as large as 130,000 daltons.     

Stable low molecular weight RNA analyzed by staircase electrophoresis, a molecular signature for both prokaryotic and eukaryotic microorganisms

Low-molecular weight RNA (LMW RNA) analysis using staircase electrophoresis was performed for several species of eukaryotic and prokaryotic microorganisms. According to our results, the LMW RNA profiles of archaea and bacteria contain three zones: 5S RNA, class 1 tRNA and class 2 tRNA. In fungi an additional band is included in the LMW RNA profiles, which correspond to the 5.8S RNA. In archaea and bacteria we found that the 5S rRNA zone is characteristic for each genus and the tRNA profile is characteristic for each species. In eukaryotes the combined 5.8S and 5S rRNA zones are characteristic for each genus and, as in prokaryotes, tRNA profiles are characteristic for each species. Therefore, stable low molecular weight RNA, separated by staircase electrophoresis, can be considered a molecular signature for both prokaryotic and eukaryotic microorganisms. Analysis of the data obtained and construction of the corresponding dendrograms afforded relationships between genera and species; these were essentially the same as those obtained with 16S rRNA sequencing (in prokaryotes) and 18S rRNA sequencing (in eukaryotes).     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Ribosomal internal transcribed spacer 2 (ITS2) exhibits a common core of secondary structure in vertebrates and yeast.

Molecular mechanisms of ITS2 processing, a eukaryotic insertion between the 5.8S and LSU rRNA, remain largely elusive even in yeast. To delineate ITS2 structural and functional features which could be common to eukaryotes, we first produced phylo-genetically supported folding models in the vertebrate lineage, then tested them in deeper branchings and, more particularly, among yeasts. ITS2 comparisons between four Teleostei, a Chondrichthyes specimen and two jawless organisms have revealed a common folding architecture in four to five domains of secondary structure emerging from a preserved structural core. This folding, largely reminiscent of ITS2 architecture in mammals, is also preserved in amphibia and in chicken, despite dramatic sequence variations. Preferential conservation is located around a central loop and at the apex of a long stem in the ITS2 3'-half. Interestingly, these two independent structural features contain, respectively, the 3'-ends of the two transient rRNA precursors 8S and 12S RNA identified in mammals, suggesting a preservation of these intermediates of processing over the entire vertebrate group. Surprising similarities between the vertebrate ITS2 folding shape and that of invertebrates as well as protista have made intriguing the significant differences from the yeast model. A detailed comparative analysis including four relatively close species and Schizosaccharomyces pombe, a deep yeast branching, has revealed an alternative phylogenetically supported four-domain folding presenting strong similarities to the vertebrate model. Remarkably, the two best conserved regions of vertebrates have unambiguously preserved counterparts which are also sites for internal processing in yeast. Therefore, molecular mechanisms involved in ITS2 excision in vertebrates and yeast might be more closely related than currently believed and might require a very similar trans -acting machinery.     

Cell-free synthesis of mouse corticotropin. Evidence for two high molecular weight gene products.

Polysomes or mRNA prepared from cultured AtT-20/D16v mouse pituitary adenocarcinoma cells direct the efficient incorporation of amino acid into newly synthesized material in the presence of wheat germ translational factors. A significant franction of the total cell-free product is specifically immunoprecipitable with corticotropin antibody purified by immune affinity chromatography. Analysis of the cell-free synthesized immunoreactive products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that two high molecular weight corticotropin species (Mr congruent to 32,500 and 28,000) are synthesized in an approximate 2:1 ratio. Neither product contains carbohydrate based upon concanavalin A chromatography or exposure to polysaccharidases. The smaller molecular weight product does not appear to arise from proteolytic processing since both species are synthesized in approximately the same ratio in cell-free reaction mixtures directed by either polysomes or mRNA. These results suggest that AtT-20/D16v cells contain two distinct mRNA poluations specifying the synthesis of two different high molecular weight forms of mouse corticotropin.     

Effect of alkylation on the physical properties of simian virus 40 T-antigen species.

We analyzed large and small species of T-antigen by immunoprecipitation and two-dimensional gel electrophoresis. The T-antigen species were subjected to electrophoresis either directly or after reduction and alkylation with N-ethylmaleimide. Treatment with N-ethylmaleimide improved the resolution of large-T by two-dimensional gel electrophoresis and was a requirement for the resolution of small-t antigen on two dimensional gels. Large-T did not form a discrete protein spot, but rather formed a streak from approximately pH 6.5 to 6.9 on isoelectric focusing gels. Small-t formed a sharp protein spot at approximately pH 7.2 when subjected to electrophoresis under non-equilibrium conditions which extended the pH gradient to include proteins with basic isoelectric points. Treatment with N-ethylmaleimide decreased the mobility of the T-antigen species during sodium dodecyl sulfate gel electrophoresis. We suggest that the apparent increase in molecular weight was due to the association of N-ethylmaleimide with cysteine-rich regions of these proteins. Viable deletion mutants of simian virus 40 which do not induce the synthesis of small-t but product small-t-related polypeptides were used to localize the cysteine-rich region of small-t to between 0.54 and 0.59 on the genetic map of simian virus 40.     

Unusual ribosomal RNA of the intestinal parasite Giardia lamblia.

The anaerobic protozoan Giardia lamblia is a common intestinal parasite in humans, but is poorly defined at molecular and phylogenetic levels. We report here a structural characterization of the ribosomal RNA (rRNA) and rRNA genes of G. lamblia. Gel electrophoresis under native or non-denaturing conditions identified two high molecular weight rRNA species corresponding to the 16-18S and 23-28S rRNAs. Surprisingly, both species (1300 and 2300 nucleotides long, respectively) were considerably shorter than their counterparts from other protozoa (typically 1800 and 3400 nucleotides), and from bacteria as well (typically 1540 and 2900 nucleotides long). Denaturing polyacrylamide gel electrophoresis identified a major low molecular RNA of 127 nucleotides and several minor species, but no molecules with the typical lengths of 5.8S (160 nucleotides) and 5S (120 nucleotides) rRNA. The G. lamblia 1300, 2300, and 127 nucleotide RNAs are encoded within a 5.6 kilobase pair tandemly repeated DNA, as shown by Southern blot analysis and DNA cloning. Thus, the rRNA operon of this eukaryotic organism can be no longer than a typical bacterial operon. Sequence analysis identified the 127 nucleotide RNA as homologous to 5.8S RNA, but comparisons to archaebacterial rRNA suggest that Giardia derived from an early branch in eukaryotic evolution.     

Interspecific variations in proteins synthesized by mammalian mitochondria.

The products of mitochondrial protein synthesis in established cell lines of various mammalian species were labelled with [35S]methionine and their number and apparent molecular weights determined by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis and fluorography. Proteins synthesized by isolated rat liver mitochondria were labelled with [3H]valine and similarly characterized. Each species had a distinctive pattern of from 10 to 13 mitochondrially synthesized proteins with apparent molecular weights between 10,000 and 50,000. No differences were detected in the number or electrophoretic mobility of the mitochondrially synthesized proteins of SV-40-transformed and nontransformed WI-38 cells.     

Identification and differentiation of Cryptosporidium species by capillary electrophoresis single-strand conformation polymorphism

Cryptosporidium species generally lack distinguishing morphological traits, and consequently, molecular methods are commonly used for parasite identification. Various methods for Cryptosporidium identification have been proposed, each with their advantages and disadvantages. In this study, we show that capillary electrophoresis coupled with single-strand conformation polymorphism (CE-SSCP) is a rapid, simple and cost-effective method for the identification of Cryptosporidium species and genotypes. Species could be readily differentiated based on the SSCP mobility of amplified 18S rRNA gene molecules. Clones that differed by single-nucleotide polymorphisms could be distinguished on CE-SSCP mobility. Profiles of species known to have heterogenic copies of 18S rRNA gene contained multiple peaks. Cloning and sequencing of Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium fayeri and Cryptosporidium possum genotype 18S rRNA gene amplicons confirmed that these multiple peaks represented type A and type B 18S rRNA gene copies. CE-SSCP provides a reliable and sensitive analysis for epidemiological studies, environmental detection and diversity screening.     

Ribosomal ribonucleic acids of cultured cells: A preliminary survey of differences among mammalian species detectable by polyacrylamide gel electrophoresis

Summary Ribosomal ribonucleic acids (rRNA's) of cultured cells from various species were compared by polyacrylamide gel electrophoresis. Electrophoretic mobility of the 28 S RNA component varied according to species. Human cell 28 S rRNA was distinguishable from that of monkey cells. The mobility of chimpanzee 28 S rRNA was identical to that from human cells, but different from that of monkey cells. Cat, goat, and swine cell 28 S rRNA's were distinguishable from one another and from both human and monkey cell rRNA's. The mobilities of bovine, rabbit, rat, hamster, and mouse cell 28 S rRNA's were virtually identical to that from monkey cells. No apparent correlation existed between the mobility of 28 S rRNA and the currently accepted phylogenetic position of the donors. Normal cells, tumor cells, and virus-infected cells from the same species did not differ, but in some instances embryonic and adult cells were distinguishable. The 18 S rRNA components did not show variations comparable to those of the 28 S rRNA components. This work was supported by the Office of Naval Research and the Bureau of Medicine and Surgery, United States Navy, under a contract between the Office of Naval Research and the Regents of the University of California.     

Gel electrophoretic fractionation of RNAs by partial denaturation with methylmercuric hydroxide.

The changes in agarose gel electrophoretic velocities of several RNAs of varying molecular weight and base composition with concentration of the denaturant, methylmercuric hydroxide (MMH), have been studied. At intermediate MMH concentrations, the mobility of any one species is intermediate between the “native” (no MMH) and fully denatured (5 mm MMH) values. A + U-richer RNAs are partially denatured at lower concentrations of MMH than are G + C-richer RNAs. Electrophoresis at intermediate MMH concentrations is useful for resolving some RNA species that are not well resolved either in the absence of MMH or under fully denaturing (5 mm MMH) conditions. It is shown that it is possible to carry out MMH electrophoresis in polyacrylamide gels; this is useful for low molecular weight RNAs. An equation to correlate changes in mobility between the native and denatured states is proposed.     

A comparative study of tropomyosin from vertebrate ventricles

A comparative study of vertebrate ventricle tropomyosin has been carried out from the viewpoint of molecular evolution. The ventricles containing one-component tropomyosin were generally known, and in this paper those containing two components were also found in 8 species among mammals, reptiles, amphibia, and fish, but not among birds. The two components were concluded to be authentic tropomyosin and not artifacts since they showed lower electrophoretic mobilities in the presence of urea, and they were precipitated at pH 4.5 and bound to F-actin. Studies on cysteine contents and cyanogen bromide cleavage peptide patterns revealed that the characteristics of the two tropomyosin components from pig, turtle, amphibia and carp ventricles varied increasingly in that order from typical alpha- and beta-characteristics as seen in rabbit skeletal muscle tropomyosin. The single component of chicken ventricle tropomyosin showed alpha component characteristics in its electrophoretic mobility and cysteine content, and beta component characteristics in cyanogen bromide cleavage peptide pattern. The two components of carp ventricle tropomyosin seemed to be the most primitive, having two cysteine residues per molecule and a cyanogen bromide cleavage peptide pattern different from those of the two components of rabbit skeletal muscle.