Multiple Regulatory Events in the Development of Competence for Genetic Transformation in Haemophilus influenzae

Cyclic adenosine 3',5'-monophosphate derepresses enzyme synthesis but does not yield high levels of transformability or detectable levels of competence-related envelope polypeptides. Additional regulatory events must trigger complete expression of competence.     

A new class of soluble basic protein precursors of the cornified envelope of mammalian epidermis

The cornified envelope hs been shown to be formed beneath the plasma membrane as a result of the cross-linking of soluble and membrane-associated precursor proteins by transglutaminase. We have obtained a monoclonal antibody which reacts with the periphery of cells in the upper layers of human epidermis by indirect immunofluorescence (IIF) following immunization of mice with cornified envelopes of cultured human keratinocytes. The antibody also stained the cell peripheries of bovine, rat and mouse epidermis as well as stratified epithelium. Neutral buffer extracts of human cultured keratinocytes and epidermis examined under denaturing conditions contained polypeptides of molecular weight 14 900 and 16 800 which reacted with the antibody, and an additional component of molecular weight 24 800 was found in cultured cells. The polypeptides were shown to have a pI of about 9.0. Under non-denaturing conditions the two lower-molecular-weight polypeptides had an apparent molecular weight of 30 000, while the 24 800 protein had one of 60 000. Incubation of the polypeptides under conditions that activate transglutaminase resulted in a disappearance of the polypeptides or the formation of cross-linked products. Basic polypeptides with somewhat different pI values and molecular weights were identified in neutral buffer extracts of bovine and rat epidermis. The HCE-2 antibody appears to identify a new class of basic protein precursors of mammalian cornified envelope.     

Envelope synthesis during the cell cycle in Escherichia coli B/r

The rate of synthesis of envelope proteins and phospholipids during the cell cycle of Escherichia coli B/r has been studied using both synchronous cultures and random cultures, first labelled and then subsequently fractionated on an age basis by the membrane elution technique. The rate of total protein synthesis and of phospholipid synthesis, measured by incorporation of [2-³H]glycerol into whole cells, was found to increase exponentially throughout the cell cycle. Total envelope protein was also synthesized continuously throughout the cycle, but the rate of synthesis showed a stepwise pattern with a discrete doubling in rate in the first half of the cycle. Analysis of the pattern of synthesis of about 29 individual envelope polypeptides by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography revealed that the great majority followed the pattern of the bulk measurements, with a discrete increase in rate of synthesis early in the cycle. One envelope polypeptide, molecular weight 76,000, was, however, only synthesized during a brief period, near the time of division of the bacteria. Pulse-chase studies of envelope polypeptide synthesis in synchronous cultures demonstrated that (1) synthesis and insertion of polypeptide into the envelope was always completed within the pulse period; (2) no post-synthetic modification of polypeptides was detected; (3) one group of polypeptides, including a major outer membrane protein, maintained a stable association with the envelope, whilst a second group displayed considerable “turnover”; (4) about 70% of newly synthesized 76,000 molecular weight protein was lost from the envelope during the succeeding generation.     

Rapid metabolism of moloney leukemia virus precursor polypeptides in virus infected Swiss mouse embryo cells.

Viral protein synthesis in Moloney murine leukemia virus infected high passage mouse embryo cells was studied utilizing monospecific antisera to the viral core protein p30 and envelope protein gp71. Pulse-chase analysis of [³⁵S]methionine-labeled polypeptides in combination with the demonstration of the presence of either gp71 or p30-specific antigenic determinants in them indicated a 84,000-dalton polypeptide as the precursor of viral glycoproteins and four metabolically unstable polypeptides of approximate molecular weights 88,000, 72,000, 62,000, and 39,000 as the precursors of viral core protein, p30. The p30-containing 88,000 and 72,000-dalton polypeptides were distinctly seen in this system under normal growth conditions. Further, the processing of p30 precursors was very rapid and was complete during a 40 min chase while only partial processing of glycoprotein precursor was observed during the same period.     

Proteins of the cell envelope of a marine pseudomonad, Pseudomonas BAL-31

The protein composition of the envelope fraction of Pseudomonas BAL-31 was studied by polyacrylamide gel electrophoresis. Two major polypeptides of molecular weights 130 000 and 110 000 were found. These two polypeptides, which account for as much as 40–50% of the total protein of the envelope, are associated with the outer membrane. One of these proteins might be a glycoprotein. The inner membrane contains a more heterogeneous collection of smaller polypeptides.     

Cell-Free Synthesis of the D2-Cell Adhesion Molecule: Evidence for Three Primary Translation Products

The D2-cell adhesion molecule (D2-CAM) is a membrane glycoprotein that is involved in cell-cell adhesion in the nervous system. To study the biosynthesis of D2-CAM we have translated free and membrane-bound polysomes from rat brain in vitro in the rabbit reticulocyte lysate system. D2-CAM was exclusively synthesized on membrane-bound polysomes. The primary translation products of D2-CAM were three polypeptides of apparent molecular weights 187,000, 134,000, and 112,000. No interconversion between these polypeptides was detected. In contrast to previous suggestions, we conclude that all three D2-CAM polypeptides are primary translation products. When translating polysomes from embryonic and postnatal rat brain, we found that the relative amounts of the three polypeptides synthesized varied with age. Their molecular weights, however, were not age-dependent.     

Origin of mitochondrial enzymes. V. The polypeptide character and the biosynthesis of rat liver cytochromec oxidase polypeptides by mitochondria

Isolated rat liver mitochondria were labeled in vitro withl-[¹⁴C]leucine. Sixty percent of the incorporated radioactivity was found to reside in subunits 1, 2, and 3 of cytochromec oxidase with apparent molecular weights of approximately 33,000, 25,000, and 20,000, respectively. The results indicate that these are the predominant products of protein synthesis under the conditions employed. The enzyme complex, as derived by immunoprecipitation, was found to contain four additional polypeptides with apparent molecular weights of 17,000, 12,500, 7000, and 3500. A comparison of electrophoretic profiles of the rat liver and beef heart enzyme reveals that the apparent molecular weights of all polypeptides are remarkably similar.To be submitted as partial fulfillment of the requirements for the Ph.D. degree of this institution.     

Cell-free synthesis of herpes simplex virus proteins.

Polyribosomes isolated from herpes simplex virus type I (HSV-1)-infected cells have been used to program a eucaryotic cell-free translation system. At least 10 HSV-specific polypeptides, with apparent molecular weights of 25,000 to 160,000, are synthesized by wild-type HSV-infected polyribosomes. Polyribosomes prepared from thymidine kinase-negative mutants of HSV direct the synthesis of three putative nonsense termination polypeptides. HSV-specific polypeptides synthesized in vitro are precipitated with antiserum to HSV-infected cell proteins.     

Polypeptides of a Light-Harvesting Complex of the Diatom Phaeodactylum tricornutum Are Synthesized in the Cytoplasm of the Cell as Precursors

A light-harvesting fucoxanthin-chlorophyll a/c-protein complex has been isolated from the diatom Phaeodactylum tricornutum by detergent extraction of thylakoid membranes coupled with sucrose density gradient centrifugation. The isolated complex was devoid of photochemical activity and displayed spectral characteristics consistent with light harvesting function. It has three major polypeptides of apparent molecular weights 18,000, 19,000, and 19,500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Using protein synthesis inhibitors, these polypeptides were shown to be synthesized on 80S cytoplasmic ribosomes. Antibodies raised to a mixture of the 19,000 and 19,500 dalton components of the complex were used to demonstrate structural similarity among the three polypeptide components. Immunoprecipitation from primary translation products synthesized in a reticulocyte lysate system primed with P. tricornutum poly(A) RNA, indicates that the polypeptide components are synthesized as precursors 3,000 to 5,000 daltons larger than the mature polypeptides.     

Biosynthesis of alpha-fetoprotein in cultured hepatoma cells

Pulse and pulse-chase experiments demonstrated that a heterogeneous polypeptide with an apparent Mr = 68,000 was the first intracellular anti-alpha-fetoprotein (AFP)-precipitable polypeptide synthesized by rat Mc-A-RH-7777 hepatoma cells. The 68,000-dalton polypeptide may consist of polypeptides with apparent molecular weights ranging from 68,000 to 70,000. It was the precursor of two intracellular anti-AFP-precipitable polypeptides of 69,000 and 73,000 apparent molecular weight. The latter were secreted into the medium without further processing. The anti-AFP-precipitable polypeptides in both cells and medium incorporated [3H]glucosamine, indicating that these polypeptides are at least partially glycosylated. The 68,000-dalton polypeptide in cells was bound mostly to concanavalin A-Sepharose, whereas the 69,000-dalton polypeptide was entirely unbound. The 73,000-dalton polypeptide consisted of concanavalin A-bound and -unbound variants. Tunicamycin completely abolished the uptake of [3H]glucosamine into anti-AFT-precipitable polypeptides in both cells and medium, and the resulting polypeptide of apparent Mr = 66,000 did not bind to concanavalin A-Sepharose. Tunicamycin did not affect the synthesis or secretion of AFP by hepatoma cells.     

Biosynthesis and degradation of storage protein in spores of the fungus Botryodiplodia theobromae

Muiridin, a spore-specific protein of the fungus Botryodiplodia theobromae, comprises about 25% of the mature pycnidiospore protein. It has an apparent molecular weight of 16,000 to 17,000 and is rich in glutamine, asparagine, and arginine. Muiridin is synthesized in developing spores via a precursor with an apparent molecular weight of 24,000. Two other polypeptides present in young developing spores with apparent molecular weights of 18,000 and 15,000 are immunologically related to muiridin. We propose a pathway for muiridin synthesis. Muiridin is actively degraded during the germination of spores from 30-day-old cultures. This degradation is independent of exogenous amino acids in the germination medium. In contrast, glutamine and, to a lesser extent, asparagine partially inhibit the degradation of muiridin during germination of spores from 7-day-old cultures.     

Regulation of accumulation of the major thylakoid polypeptides in Chlamydomonas reinhardtii y-1 at 25 degrees C and 38 degrees C

The amount of messenger RNA (mRNA) for polypeptides of the chlorophyll a/b-protein complex of thylakoid membranes in etiolated and greening cells of Chlamydomonas reinhardtii y-1 was examined by immunoprecipitation and electrophoresis of products of in vitro translation to determine at which stage production of these polypeptides is regulated. Cells grown 4 d in the dark at 25 degrees C contained small amounts of translatable mRNA for the major membrane polypeptides. Exposure of these etiolated cells to light, under conditions in which the membrane polypeptides accumulated, resulted in a significant increase in the quantity of the mRNA. In contrast, when etiolated cells were incubated for 1-2 h in the dark at 38 degrees C, translation assays indicated that mRNA for the membrane polypeptides became abundant. Moreover, the quantity of the mRNA did not increase when these cells subsequently were exposed to light. Therefore, at 38 degrees C the cellular level of the polypeptides is not regulated by synthesis of mRNA. The in vitro synthesized polypeptides, which were precipitated with antibodies prepared against the purified thylakoid polypeptides, had apparent molecular weights of 31,500 and 30,000. The corresponding immunoprecipitated polypeptides made in vivo had apparent molecular weights of 29,500 and 26,000. Thus, the membrane polypeptides are made as precursors. No net accumulation of the polypeptides occurred in cells in the dark at 38 degrees C, but immunoreactive polypeptides the size of the mature membrane components were labeled during incubation of cells with [14C]acetate in the dark. These results indicated that the mRNA was translated in the dark, but since the polypeptides did not accumulate, the products of translation were probably degraded. We conclude from our experiments that at 25 degrees C production of the polypeptides is regulated by the level of translatable mRNA in the cells. At 38 degrees C, however, the accumulation of the polypeptides is controlled by posttranslational processes.     

Protein synthesis during the transition from the resting to the growing state in suspension cultures of Paul's Scarlet rose cells

Cells derived from Paul's Scarlet rose ( Rosa sp. ) were grown in the chemically defined medium of Nesius. When a stationary phase culture was diluted with fresh medium, growth was initiated after a pronounced lag period. DNA replication, as revealed by thymidine labeling and autoradiography, did not begin until 36 h, and mitotic figures were not observed until 48 h after dilution. A 10–15 fold increase in the rate of protein synthesis occurred during the lag period. This was brought about by a 3.5 fold increase in the amount of ribosomal RNA per cell, plus a doubling of both the percentage of ribosomes that are present as polyribosomes and the average number of ribosomes per polyribosome. The spectrum of polypeptides synthesized by these cells during the lag and early log periods of growth was examined. Polyribosomes were extracted from the cells at intervals preceding and accompanying the initiation of proliferative growth. The polyribosomes were translated in a wheat germ cell-free protein synthesizing system and the ³⁵S-methionine-labeled translation products were separated on polyacrylamide slab gels and by 2-dimensional gel electrophoresis. Comparatively few differences were observed between stationary phase, lag phase and log phase cells in terms of the spectrum of polypeptides synthesized in vitro. However, these various phases of the growth cycle could be characterized by a relatively high rate of synthesis of a few specific polypeptides. That is, while most proteins are synthesized throughout the growth cycle and even in non-growing cells at approximately the same relative rates, there are a few variable proteins whose synthesis marks a particular phase of the growth cycle.     

Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS- polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000- 75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.     

Detection and characterization of GTP-binding proteins in the chloroplast envelope of spinach (Spinacia oleracea)

Proteins binding guanosine triphosphate (GTP) have emerged as important regulators in several cellular processes in plants. To investigate any role of such proteins in chloroplast functions, we subjected envelope, stroma and thylakoid fractions isolated from spinach chloroplasts to two different GTP-binding assays. With both methods, we detected GTP-specific binding only in the envelope fraction. Two chloroplast envelope proteins with the apparent molecular weights of 30.5 and 33.5 kDa, respectively, bound [α-³²P]GTP after SDS-PAGE followed by electroblotting onto a PVDF-membrane and renaturation. Both proteins were intrinsic proteins located in the outer chloroplast envelope. Also, when the fractions were incubated with [α-³²P]GTP, followed by periodate oxidation and borohydride reduction to cross-link GTP to proteins, two proteins in the envelope fraction, of apparent molecular weights of 28 and 39 kDa, appeared to specifically bind GTP. When agents that stimulate heterotrimeric G-proteins, cholera toxin or the mastoparan analogue mas7, were added to isolated chloroplast envelope, the binding of radiolabelled GTP to the 39 kDa protein, a protein of the inner chloroplast envelope, was stimulated, whereas GTP-binding of the 28 kDa protein, a protein of the outer envelope, was unchanged. Mas7 also stimulated synthesis of monogalactosyl diacylglycerol in isolated chloroplast envelope. The occurrence and regulation of GTP-binding proteins in the chloroplast envelope suggests that GTP-binding proteins could be involved in communication with the extraplastidic compartment during chloroplast biogenesis and development.     

Structural Components of Oriboca Virus

Analysis of purified Oriboca virions by neutral, sodium dodecyl sulfate polyacrylamide-gel electrophoresis indicated the presence of three structural polypeptides designated V-1, V-2, and V-3 on the basis of their relative electrophoretic mobilities in 8% gels. Polypeptides V-2 and V-3 are glycopeptides associated with the virion envelope as demonstrated by the preferential incorporation of labeled glucosamine into the polypeptides and by release of the polypeptides from the intact virion by the nonionic detergent NP-40. Polypeptide V-1 is the protein component of the nucleoprotein core of Oriboca virus as evidenced by the specific incorporation of uridine into the nucleoprotein, its release from the intact virion by NP-40 treatment, and its separation by both rate-zonal and isopycnic density gradient centrifugation from both the intact virion and envelope components. Molecular weights have been tentatively assigned to the polypeptides by extrapolation from the structural polypeptides of Sindbis virus when both are run in the same gel. Polypeptide V-1 has an apparent molecular weight of 20,000 to 23,000; V-2, 30,000 to 32,000; and V-3, 83,000 to 85,000.     

Subchloroplast localization of polypeptides synthesized by chloroplasts during senescence

To study the localization of polypeptides synthesized by isolated senescent chloroplasts we have fractionated the chloroplasts into stroma, envelope and thylakoid components. The validity of the fractionation procedure was tested by assaying both chlorophyll and enzyme markers, as well as the polypeptide composition of each fraction. Plastids in the transition of etioplast to chloroplast, senescent chloroplasts and kinetin-treated chloroplasts produced acceptable fractions, although their polypeptide compositions varied considerably during the ontogeny, particularly those of the envelope. Most of the polypeptides synthesized by isolated senescent chloroplasts were incorporated into the thylakoids except for a 58 kDa polypeptide localized in the stroma and some minor polypeptides present in both stroma and envelope. Although most of the polypeptides synthesized by isolated chloroplasts from kinetin-treated leaves were incorporated into the thylakoid membrane, several polypeptides were found in the stroma (90, 80, 65 and 54 kDa) and in the envelope (100, 75, 48 and 28–30 kDa). The results indicate that early in senescence, the polypeptides of the envelope change but, that probably, most of the new polypeptides are synthesized in the cytoplasm.     

Radiolabelling of DNA/polypeptide complexes in isolated bulk DNA and in residual nuclear matrix DNA by nick-translation

Conditions are described that allow 32P-radiolabelling and detection of tight complexes between DNA and polypeptides by nick-translation. Prolonged nick-translation of purified bulk DNA results in radiolabelled complexes migrating on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 54 kd respectively. Residual nuclear matrix DNA which is not accessible to DNase I on the nuclear level becomes accessible to radiolabelling by nick-translation on the nuclear matrix level. In this case the in situ radiolabelled complexes migrate on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 100 kd. The DNA/polypeptide complexes are stable during treatments with SDS, beta-mercapto ethanol and alkali which points to covalent bonds between the polypeptides and DNA strands.