Oxygen concentration, nitrogenase activity and heterocyst frequency in the leaf cavities of Azolla filiculoides Lam

Following exposure to UV light the synthesis of six polypeptides (112, 100, 89, 76, 71 and 65 kDa) was found to be enhanced or induced in the alga Chlamydomonas reinhardtii. Treatment with 4-nitroquinoline-N-oxide (NQO) resulted in the enhanced/induced synthesis of six polypeptides with molecular masses similar to those enhanced following exposure to UV light. Heat shock resulted in the enhanced synthesis of five polypeptides (89, 76, 71, 60 and 22 kDa), three of which (89, 76 and 71 kDa) had apparently identical mobilities to polypeptides whose synthesis was enhanced following UV treatment.     

Proteins Associated with Adaptation of Cultured Tobacco Cells to NaCl

Cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) adapted to grow in medium containing high levels of NaCl or polyethylene glycol (PEG) produce several new or enhanced polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intensities of some of the polypeptide bands (molecular weights of 58, 37, 35.5, 34, 26, 21, 19.5, and 18 kilodaltons) increase with increasing levels of NaCl adaptation, while the intensities of other polypeptide bands (54, 52, 17.5, and 16.5 kilodaltons) are reduced. Enhanced levels of 43- and 26-kilodalton polypeptides are present in both NaCl and PEG-induced water stress adapted cells but are not detectable in unadapted cells. In addition, PEG adapted cells have enhanced levels of 29-, 17.5-, 16.5-, and 11-kilodalton polypeptides and reduced levels of 58-, 54-, 52-, 37-, 35.5-, 34-, 21-, 19.5-, and 18-kilodalton polypeptide bands.

Synthesis of 26-kilodalton polypeptide(s) occurs at two different periods during culture growth of NaCl adapted cells. Unadapted cells also incorporate ³⁵S into a 26-kilodalton polypeptide during the later stage of culture growth beginning at midlog phase. The 26-kilodalton polypeptides from adapted and unadapted cells have similar partial proteolysis peptide maps and are immunologically cross-reactive. During adaptation to NaCl, unadapted cells synthesize and accumulate a major 26-kilodalton polypeptide, and the beginning of synthesis corresponds to the period of osmotic adjustment and culture growth. From our results, we suggest an involvement of the 26-kilodalton polypeptide in the adaptation of cultured tobacco cells to NaCl and water stress.

     

Large-scale purification of hnRNP proteins from HeLa cells by affinity chromatography on ssDNA-cellulose

A purification procedure for proteins which bind heterogeneous nuclear RNA (hnRNP proteins) is described. The procedure, which entails standard chromatographic fractionations (single-stranded DNA cellulose, hydroxyapatite) and detection with specific antibodies, allows a large-scale preparation of these proteins and the partial separation of different polypeptides. By this method, polypeptides of higher molecular mass (53-55 kDa) can be purified, which are structurally and antigenically related to the 'canonical' hnRNP core proteins (34-43 kDa) that constitute the 40S hnRNP complexes. We also show that HeLa cells contain a protease that cleaves hnRNP core proteins to discrete smaller polypeptides of 22-28 kDa. Such protease, which has been partially purified, appears to copurify extensively with some of the hnRNP proteins.     

A function for pericardial cells in an insect

The pericardial cells (PCs) of fifth instar Calpodes ethlius larvae are functionally adapted for filtering hemolymph and sequestering and digesting proteins. They also have a structure appropriate for the synthesis of proteins for secretion. PC secretion has been investigated by labelling the cells with [³⁵S]methionine ti vitro with detection of newly synthesized polypeptides appearing in the medium by electrophoresis and fluorography. Sources possibly contributing to the appearance of newly synthesized polypeptides in the medium, such as cell breakdown and fat body contamination have been ruled out. The post-incubation medium of PCs contains at least six newly synthesized polypeptides. Three of these polypeptides, having relative molecular masses of 82, 57 and 43 kDa, react with antibodies to hemolymph. At least one additional polypeptide is similar by two-dimensional analysis to that naturally present in hemolymph. PCs incubated together with the heart to which they are normally attached, secrete additional polypeptides that are presumed to come from the heart. The 82 kDa polypeptide secreted by the PCs is similar to the subunits of arylphorin secreted by fat body and other tissues. We conclude that PCs secrete proteins into the hemolymph although the amount may be small relative to that of the fat body.     

The purification of a human mismatch-binding protein and identification of its associated ATPase and helicase activities.

A mismatch-binding protein has been purified an estimated 4500-fold from HeLa nuclear extracts using four different chromatographic steps. Two polypeptides of apparent molecular weight of 160,000 and 100,000 were present in the final affinity-purified fraction as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial proteolytic clipping of the protein-DNA complexes visualized after UV treatment indicated that the 100-kDa polypeptide is most likely a degradation product of the 160-kDa polypeptide. UV cross-linking experiments have shown that both these polypeptides bind specifically to oligonucleotide duplexes containing G/T mismatches. Direct DNA binding studies and band-shift competition assays showed that although the mismatch-binding protein binds with highest affinity to oligonucleotides containing G/T mismatches, it is also capable of binding to oligonucleotides containing other mispairs. The purified protein has an associated Mg(2+)-dependent ATPase activity, which is markedly enhanced in the presence of single-stranded DNA. A helicase capable of unwinding a 34-mer oligonucleotide, annealed to a complementary sequence in single-stranded M13, also copurified with the mismatch-binding protein. This reaction occurs in an ATP- and magnesium-dependent manner.     

Changes in the polypeptide patterns of barley seedlings exposed to jasmonic Acid and salinity

Soluble and thylakoid membrane proteins of jasmonic acid (JA)-treated and salt-stressed barley (Hordeum vulgare L.) seedlings were investigated using 15% sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. High JA concentrations induced marked quantitative and qualitative changes in polypeptide profiles concerning mainly the proteins with approximately equal mobility, as in NaCl-stressed plants. The most obvious increase in thylakoid polypeptide band intensity was at 55 to 57 kilodaltons (kD). The relative share of some polypeptides with apparent molecular masses above 66 kD and of polypeptides with lower molecular masses in the region of 20.5 to 15 kD was enhanced. At the same time, one new band at 31 to 31.5 kD was well expressed at 25 and 250 micromolar JA concentrations and became discernible in the 100 micromolar NaCl-treated plants. The intensity of some polypeptides of soluble proteins (molecular masses of 60, 47, 37, 30, and 23.4 kD) increased with increasing JA concentration, whereas the intensities of other polypeptide bands (55, 21.4, and 15 kD) decreased. Enhanced levels of 60-, 47-, 34-, and 30-kD polypeptides and reduced levels of 55- and 15-kD polypeptides were present in NaCl-treated plants. The appearance of one new polypeptide, of 25.1 kD, was observed only in NaCl-treated plants. At 100 millimolar NaCl, an eightfold increase in proline content was observed while at 250 micromolar JA, the proline content was threefold over the control. It is hypothesized that exogenously applied jasmonates act as stress agents. As such, they provoke alterations in the proline content and they can modulate typical stress responses by induction of stress proteins.     

Comparative analysis of proteins from the fibrous sheath and outer dense fibers of rat spermatozoa

The protein composition of the fibrous sheath (FS) and the outer dense fibers (ODF), two cytoskeletal components of the tail of spermatozoa, was compared by using polyacrylamide gel electrophoresis and immunochemistry applied to Western blots and to spermatozoa. Isolated FS and ODF, the purity of which were verified by electron microscopy (EM), were denatured and either run on sodium dodecyl sulfate-polyacrylamide gels or used to raise antibodies. The gels revealed at least 18 and 14 polypeptide bands for the FS and ODF, respectively. The major bands of the FS had molecular masses of 75, 27.5, and 14.4 kDa, whereas the major bands of the ODF-connecting piece had molecular masses of 32-26, 20, 14.4, 84, and 80 kDa. Several prominent FS and ODF bands were found to comigrate on gels, and the 14.4 kDa polypeptides had similar electrophoretic properties. Anti-FS serum reacted with the majority of Western blot-transferred FS polypeptides, but also cross-reacted strongly with a major 14.4 kDa ODF polypeptide and with less affinity to other major ODF polypeptides. Anti-ODF serum reacted with the majority of ODF polypeptides, but also cross-reacted strongly with a major 14.4 kDa FS polypeptide, and with less affinity to several other FS polypeptides including the 75 kDa band. Antibodies affinity-purified from the 14.4 kDa FS polypeptide only cross-reacted with the 14.4 kDa ODF polypeptide, whereas antibodies purified from the 14.4 kDa ODF polypeptide cross-reacted with 14.4, 27.5, 57, and 63 kDa FS polypeptides. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. This study demonstrates that the FS and ODF, both composed of many polypeptides, several having similar molecular weights, are related cytoskeletal structures as they have epitopes in common, and both contain 14.4 kDa polypeptides with common antigenic and electrophoretic properties.     

Purification and characterization of mammalian DNA methyltransferases by use of monoclonal antibodies

Previously, we have derived murine hybridomas producing monoclonal antibodies against DNA methyltransferase from human placenta (Kaul, S., Pfeifer, G. P., and Drahovsky, D. (1984) Eur. J. Cell Biol. 34, 330-335). One of these monoclonal antibodies, M2B10, which undergoes immune complex formation also with DNA methyltransferase from P815 mouse mastocytoma cells, was used for the immunoaffinity purification of mouse and human DNA methyltransferases. In sodium dodecyl sulfate-polyacrylamide gels and in immunoblotting studies, the immunoaffinity-purified mouse DNA methyltransferase revealed 5-6 polypeptides of molecular masses 150-190 kDa. The immunoaffinity-purified human placental DNA methyltransferase was characterized by a polypeptide of 158 kDa, presumably representing the native enzyme molecule and by polypeptides of 105-108 kDa and 50-68 kDa, probably generated by a limited proteolysis of the native enzyme molecule. The immunoaffinity-purified DNA methyltransferases preferred hemimethylated DNA substrates over unmethylated ones, and among all unmethylated substrates tested, poly[(dG-dC).(dG-dC)] had the highest methyl-accepting activity. DNA polymers of at least 90 base pairs in length were required for the binding reaction of the immunoaffinity-purified human DNA methyltransferase, and this initial binding was apparently independent of the nucleotide composition of the DNA polymer and of the presence of S-adenosyl-L-methionine.     

Changes in thylakoid polypeptide phosphorylation during membrane biogenesis in Chlamydomonas reinhardii y-1

Thylakoid polypeptide phosphorylation has been studied in vivo and in vitro during plastid differentiation in Chlamydomonas reinhardii y-1. Pulse labeling cells at different stages of greening with [³²P]orthophosphate revealed differences in the pattern of protein phosphorylation. In the early phase of greening the 44–47 kDa reaction center II polypeptides were labeled but the 22–24 kDa polypeptides of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHC) were not. Later in the greening, coinciding with the formation of the antenna of Photosystem I and membrane stacking, the converse was found. Furthermore, the 22–24 kDa polypeptides of grana lamellae were less labeled than the same polypeptides found in the corresponding stroma lamellae. Polypeptides in the molecular mass range of 32–34 kDa were phosphorylated at all stages following the onset of greening. Dark-grown cells did not incorporate ³²P in vivo or in vitro into the polypeptides present in the residual thylakoids. Similarly, cells greened in the presence of chloramphenicol, in which the synthesis of reaction centers is inhibited, showed no light-stimulated phosphorylation in vitro. However, the residual 32–34 kDa and 44–47 kDa polypeptides found in thylakoids of these cells were phosphorylated in vivo, whereas the LHC polypeptides synthesized in the presence of chloramphenicol were not. Phosphorylation of the LHC polypeptides (22–24 kDa) in these cells occurred if new reaction center polypeptides and all antennae components were formed, following removal of the inhibitor and further incubation of the cells in the light. Phosphorylation of LHC polypeptides was not resumed if active reaction centers were formed in the absence of complete restoration of all antenna components (incubation in the dark or light with addition of cycloheximide). It is concluded that phosphorylation is correlated with the thylakoid polypeptide content and organization.     

The synergistic effect of hyperthermia and ethanol on changing gene expression of mouse lymphocytes

Cultured mouse lymphocytes respond to a brief incubation at an elevated temperature (41-43 degrees C) with the new and (or) enhanced synthesis of a select group of polypeptides (known as heat-shock proteins, HSPs) having relative molecular masses of 110, 100, 90, 70, and 65 kilodaltons (kDa). Expression of these HSPs is dependent on new RNA synthesis. Because the synthesis of any particular HSP is dependent on the temperature and the length of time cells remain at a particular elevated temperature, synthesis of each HSP is not necessarily coordinated with the synthesis of the other HSPs. Cultured mouse lymphocytes treated with arsenite or ethanol exhibit new and (or) enhanced synthesis of HSPs with molecular masses of 110, 90, 70, and 65 kDa but do not exhibit enhanced synthesis of the 100-kDa HSP. Short-term concurrent exposure of mouse lymphocytes to an elevated temperature and a level of ethanol, which individually do not induce detectable HSP synthesis, results in the pronounced synthesis of HSPs similar to those seen following exposure to higher levels of either stress applied separately. Thus, in this study we demonstrate that hyperthermia and ethanol stress can act synergistically to affect a dramatic change in the gene expression of mouse lymphocytes.     

Isolation and characterization of a DNA helicase from cytosolic extracts of calf thymus

A DNA helicase has been isolated from calf thymus tissue. The enzyme was enriched from crude cytosolic extracts by batchwise chromatography on phosphocellulose, followed by 35% ammonium sulfate precipitation, and subsequent chromatography on phenyl-Sepharose, single-stranded DNA cellulose, and AcA 44 gel filtration. The DNA helicase had a Stokes' radius of about 45? and a sedimentation coefficient of 4.3 S. The most purified fractions contained three polypeptides with apparent molecular weights of 110, 65, and 34 kDa. UV crosslinking with radioactive dATP stained all three major polypeptides. The helicase catalyzed the unwinding of a DNA primer from a single-stranded DNA template in an ATP- or dATP-dependent manner. DNA unwinding was also observed with CTP or dCTP, but with reduced efficiency. The helicase translocated from 3′ to 5′ on the single-stranded template it was bound to. Relationships between this DNA helicase and other calf thymus helicases will be discussed.     

Isolation and characterization of a DNA helicase from cytosolic extracts of calf thymus

A DNA helicase has been isolated from calf thymus tissue. The enzyme was enriched from crude cytosolic extracts by batchwise chromatography on phosphocellulose, followed by 35% ammonium sulfate precipitation, and subsequent chromatography on phenyl-Sepharose, single-stranded DNA cellulose, and AcA 44 gel filtration. The DNA helicase had a Stokes' radius of about 45Å and a sedimentation coefficient of 4.3 S. The most purified fractions contained three polypeptides with apparent molecular weights of 110, 65, and 34 kDa. UV crosslinking with radioactive dATP stained all three major polypeptides. The helicase catalyzed the unwinding of a DNA primer from a single-stranded DNA template in an ATP- or dATP-dependent manner. DNA unwinding was also observed with CTP or dCTP, but with reduced efficiency. The helicase translocated from 3′ to 5′ on the single-stranded template it was bound to. Relationships between this DNA helicase and other calf thymus helicases will be discussed.     

Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Effects of 2,4-D removal on the synthesis of specific proteins by Catharanthus roseus cell cultures

Total and neosynthesized proteins of periwinkle cell suspensions (Catharanthus roseus) were first investigated in cells grown in a 2,4-D-containing medium. Analysis of total (silver-stained) proteins by two-dimensional gel electrophoresis revealed that the levels of seventeen polypeptides were altered during the growth cycle of the cells. Analysis of in vivo [³⁵S]-methionine labeled polypeptides revealed differences in the synthesis of at least 35 polypeptides. Three polypeptides with molecular masses of 30, 35 and 39 kDa appeared to be specific markers of the early stationary phase. In a second sequence of experiments, cells were grown in a 2,4-D-free medium. Alterations in protein synthesis were observed: several polypeptides were expressed earlier in the 2,4-D-starved cells than in control cells; the synthesis of at least two specific polypeptides was increased in cells grown in 2,4-D-free medium, whereas the synthesis of three other polypeptides (molecular masses 33, 34 and 52.5 kDa) was switched on in these cells. As previous studies showed that 2,4-D depletion increased the alkaloid production in C. roseus cells, the present results may suggest that these polypeptides are implicated in the regulation of the alkaloid pathway.     

Gene 26 of bacteriophage T4 basal plate. I. Two expression products of the cloned gene

We have cloned the 1.9 kb EcoRV-BglII DNA fragment with T4 genes 51, 26, and 25 into the expression plasmid pT7-5 carrying a T7 promoter. The resulting recombinant plasmid, pRR5-3, contained T4 genes 26 and 25 in the correct orientation for expression. We expressed these genes using the T7 RNA polymerase/promoter system and the synthesis of three polypeptides with the molecular masses of approximately 24, 15, and 8-9 kDa was observed. Expression of genes from the subcloned DNA fragments and from the fragments carrying deletions was studied as well and the 15 kDa protein appeared to be the product of gene 25, while 24 kDa and 8-9 kDa proteins were identified as products of gene 26. The 8-9 kDa protein was shown to be expressed from the end region of gene 26. Having analysed the proteins expressed from the fragments carrying fusion of genes 26 and 25 we supposed two products of gene 26 to be encoded by the same open reading frame.     

Rapid inhibition of protein histidine phosphorylation by UV-irradiation in Xanthomonas oryzae pv. oryzae

Abstract Exposure of Xanthomonas oryzae pv. oryzae cells to 254 nm UV radiation resulted in an alteration of protein phosphorylation. Labelling of the phosphohistidine-containing proteins with molecular masses of 81 and 32 kDa, named p81 and p32, was rapidly reduced following UV irradiation in the early exponential cells, but the decrease was not detected in mid-exponential cells. Mitomycin C, a DNA replication inhibitor, and rifampicin, a drug generally used to inhibit RNA synthesis and DNA replication, were also found to reduce the histidyl phosphorylation. However, this alteration of protein phosphorylation was not hindered by chloramphenicol treatment. A possible role for these histidyl phosphopfoteins in sensing UV light is proposed.     

Salt-induced changes in protein composition in light-grown callus of Mesembryanthemum crystallinum

The halophyte Mesembryanthemum crystallinum (ice plant) has been suggested as a model for salt-tolerance in higher plants. To investigate salt-induced changes in polypeptide patterns at the cellular level, a light-grown callus of M. crystallinum with substantial chlorophyll content, was established and the effect of NaCl on the composition of phenol-extracted protein was examined by SDS- and 2D-polyacrylamide gel electrophoresis (PAGE). SDS-PAGE showed the accumulation of five polypeptides with estimated molecular masses of 40, 34, 32, 29 and 14 kDa was enhanced by the addition of 200 m M NaCl to the culture media. The addition of ABA (10 μ M ) or mannitol (400 m M ) did not elicit the same degree of accumulation of these salt-specific proteins. These polypeptides were classified into two groups according to their course of induction: early responsive (40, 34, 29 kDa) and late-responsive (32, 14 kDa) proteins. In addition, two polypeptides (20, 18 kDa) were transiently accumulated during salt treatment. Further separation of soluble proteins by 2-D gel electrophoresis, either isoelectric focusing (IEF) or non-equilibrium pH-gradient electrophoresis (NEPHGE) followed by SDS-PAGE, showed more alterations in accumulation of polypeptides by NaCl than 1-D gel electrophoresis. Overall, levels of more than 30% of basic polypeptides, detected by NEPHGE/SDS-PAGE, were altered by 200 m M NaCl treatment, while only 10% of neutral and acidic polypeptides, detected by IEF/SDS-PAGE, were changed. The enhanced expression of these proteins by salt in cultured cells is most likely related to the cellular responses to salinity, and not to the mechanism of CAM induction in this facultative halophyte.     

The small subunits of human and mouse DNA polymerase epsilon are homologous to the second largest subunit of the yeast Saccharomyces cerevisiae DNA polymerase epsilon.

Human DNA polymerase epsilon is composed of a 261 kDa catalytic polypeptide and a 55 kDa small subunit of unknown function. cDNAs encoding the small subunit of human and mouse DNA polymerase epsilon were cloned. The predicted polypeptides have molecular masses of 59.469 and 59.319 kDa respectively and they are 90% identical. The human and mouse polypeptides show 22% identity with the 80 kDa subunit of the five subunit DNA polymerase epsilon from the yeast Saccharomyces cerevisiae. The high degree of conservation suggests that the 55 kDa subunit shares an essential function with the yeast 80 kDa subunit, which was earlier suggested to be involved in S phase cell cycle control in a pathway that is able to sense and signal incomplete replication. The small subunits of human and mouse DNA polymerase epsilon also show homology to the C-terminal domain of the second largest subunit of DNA polymerase alpha. The gene for the small subunit of human DNA polymerase epsilon (POLE2) was localized to chromosome 14q21-q22 by fluorescence in situ hybridization.     

The role of Cd-binding proteins and phytochelatins in the formation of resistance to cadmium in the cell lines of <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis>

The callus lines of Nicotiana plumbaginifolia with equal resistances to cadmium were obtained under different selective conditions, including without the inhibition of phytochelatin synthesis (the Cd-R line) and with the inhibition of a butionine sulfoximine (the Cd-Ri line). The level of phytochelatin synthesis in the Cd-R and Cd-Ri lines exceeded the control value by fivefold and twofold, respectively. Three cadmium-binding proteins with molecular masses of 41, 34, and 19 kDa were observed mainly in the control line. A common feature of both resistant lines was the expression of cadmium-binding proteins with molecular masses of 40, 34, and 19 kDa. The synthesis of relatively low-molecular cadmium-binding proteins was characteristic of the resistant lines as follows: the line Cd-R expressed the proteins with molecular masses of 12.5, 11.5, and 9 kDa, while the Cd-Ri line expressed those with molecular masses of 13 and 10 kDa. We supposed that both phytochelatins and Cd-binding proteins contribute to the resistance to cadmium in N. plumbaginifolia callus lines, and, additionally, that changes in the synthesis of low-molecular Cd-binding proteins can compensate for the lack of phytochelatins.