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Biomedical subjects

L Bogorad

Publications and source records attributed to L Bogorad.

At least 109 records · Page 6Linked to original sources
PubMed1980

A facile procedure for purifying maize chloroplast RNA polymerase from whole cell homogenates.

Two DNA-dependent RNA polymerases have been purified from homogenates of maize leaves by a relatively rapid procedure involving Sepharose 4B and DEAE-cellulose chromatography followed by resolution of two RNA polymerasies on phosphocellulose. The RNA polymerase eluting from phosphocellulose at 0.11 M (NH4)2SO4 is inhibited strongly by low levels of alpha-amanitin and possesses catalytic properties and polypeptide subunits like those of maize nuclear RNA polymerase II. The RNA polymerase eluting from phosphocellulose at 0.15 M (NH4)2SO4 resembles the RNA polymerase solubilized from isolated maize chloroplasts in many ways. This enzyme and that isolated from chloroplasts are resistant to alpha-amanitin and rifamycin-SV at high concentrations. Both RNA polymerases have virtually the same Mn2+ and Mg2+ optima, Mg2+/Mn2+ activity ratios, (NH4)2SO4 sensitivity, kinetics of UMP incorporation, and temperature optima. Electrophoresis of this phosphocellulose-purified RNA polymerase on denaturing polyacrylamide slab gels reveals 14 heavily stainable polypeptides that are identical in number and molecular mass to those from chloroplast RNA polymerase. Moreover, two-dimensional tryptic maps of the 14 polypeptides from the phosphocellulose-purified RNA polymerase are very similar to the maps of corresponding polypeptides from chloroplast RNA polymerase. Using this method, relatively large quantities (0.5 mg/kg leaves) of a form of chloroplast RNA polymerase can be prepared in a few days.

Centrifugation, Density Gradient↗
PubMed1980

Preferential transcription of cloned maize chloroplast DNA sequences by maize chloroplast RNA polymerase.

Zea mays chloroplast DNA-dependent RNA polymerase in vitro preferentially transcribes maize chloroplast DNA sequences incorporated in cloned chimeric bacterial plasmids. Preferential transcription is dependent on the presence of a 27.5-kilodalton polypeptide, the S factor, which has been purified from maize chloroplasts, and also on the template's being in the supercoiled form.

Journal Article↗
PubMed1980

Sizes, locations, and directions of transcription of two genes on a cloned maize chloroplast DNA sequence.

mRNA for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] of Zea mays is complementary to an uninterrupted 1600-base-pair-long chloroplast DNA sequence that has been mapped precisely within the 4350-base-pair-long chloroplast DNA fragment Bam 9 to which it had been traced earlier [Bedbrook, J. R., Coen, D. M., Beaton, A. R., Bogorad, L. & Rich, A. (1979) J. Biol. Chem. 254, 905-910]. An additional 1400-base-pair-long uninterrupted region that is colinear with a chloroplast RNA has been detected on Bam 9. The transcript from this region is part of a 2200-nucleotide-long RNA. The remainder of the DNA sequence for the 2200-base-pair RNA maps outside Bam 9. The 1600-base-pair LS gene and the gene for the 2200-nucleotide transcript are close to one another. They are separated by an untranscribed intercistronic "gap" about 330 base pairs long. These two closely packed genes are inverted on the chromosome-i.e., their 3' termini are at opposite ends of the untranscribed gap and they map on opposite strands.

Journal Article↗
PubMed1980

Light-induced Changes in Allophycocyanin.

Several lines of evidence indicate that allophycocyanin is the previously unidentified "phycochrome" observed in extracts of blue-green algae.Fractions containing phycoerythrin, phycocyanin, and allophycocyanin and exhibiting light-induced absorbance changes were prepared from extracts of Nostoc muscorum and Fremyella diplosiphon by isoelectric focusing. Illumination of such fractions with red light (650 nanometers) causes a reduction in absorbance at 620 nm ( approximately 1 to 2%) and an increase at 560 nm. The effect, (previously observed by Björn and Björn [1976 Physiol Plant 36: 297-304]) is reversible, upon illumination with green light (550 nm). Selective immunoprecipitation of the phycobiliproteins indicates that allophycocyanin is the photoresponsive pigment.At pH 4.0 to 4.2, allophycocyanin purified from the same algae or from Phormidium luridum exhibits a light-induced absorbance change at 620 nm, which coincides with its absorption maximum at this pH; the fluorescence emission of allophycocyanin under these conditions is at 647 nm and its S(20,w) is 2.28, compatible with an alpha(1)beta(1) polypeptide composition. At neutral pH (5.8 to 7.0), allophycocyanin aggregates have a sedimentation coefficient of 4.8 ( approximately alpha(3)beta(3)) and an additional absorption peak at 640 nm appears while that at 620 nm remains unaffected. The fluorescence emission maximum of the larger aggregate is at 667 nm and the light-induced change in its absorption is shifted to 650 nm. The effect of pH changes in the range 4.0 to 7.0 on the spectral and aggregation properties of allophycocyanin is completely reversible. Changes in pH which affect allophycocyanin aggregation have parallel effects on absorption and fluorescence maxima as well as on the light-induced absorbance changes of the biliprotein.No evidence is provided to resolve whether this phycochrome plays the role of an adaptochrome.

Journal Article↗
PubMed1979

Location of the single gene for the large subunit of ribulosebisphosphate carboxylase on the maize chloroplast chromosome.

The structural gene for the large subunit of ribulosebisphosphate carboxylase (E.C. 4.1.1.39) in Zea mays is shown to be contained within a 2500 base pair sequence of chloroplast DNA. One copy of this DNA sequence is present in each circular maize chloroplast DNA molecule. It maps approximately 30,000 base pairs from the 5' end of the closest of two sets of rRNA genes and approximately 71,000 base pairs from the other set of rRNA genes.

Carboxy-Lyases↗
PubMed1979

Peptide maps comparing subunits of maize chloroplast and type II nuclear DNA-dependent RNA polymerases.

Both one- and two-dimensional peptide mapping techniques have been used to compare the 180-kilodalton subunits from maize chloroplast and type II nuclear DNA-dependent RNA polymerases (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). Despite their similar molecular mass, these 180-kilodalton subunits are not the same. Also, two-dimensional tryptic maps of the 160-, 43-, and 28-kilodalton polypeptides from maize type II nuclear RNA polymerase and of the 140-, 42-, and 27-kilodalton polypeptides from maize chloroplast RNA polymerase show that each of these six polypeptides is unique.

Chloroplasts↗
PubMed1979

Photoreversible absorbance changes in solutions of allophycocyanin purified from Fremyella diplosiphon: Temperature dependence and quantum efficiency.

Preparations of allophycocyanin isolated from the alga Fremyella diplosiphon show light-induced optical absorbance changes that suggest the presence of a photoconvertible component [Formula: see text] similar to the algal pigments described by J. Scheibe [(1972) Science 176, 1037-1039]. At pH < 4 the allophycocyanin has an absorption maximum at 620 nm. Red illumination causes a loss of absorbance in the red, centered at 620 nm, and subsequent green illumination restores the lost absorbance. We have studied this photoconversion at temperatures between 200 K and 307 K, analyzing the results in terms of photostationary states established under red (640 nm) and green (550 nm) light. As the temperature was lowered to 260 K, the state P(r) became progressively favored; the reaction P(r) --> P(g) induced by red light was attenuated but the reaction P(g) --> P(r) induced by green light was not. Decreasing the temperature from 260 K to 200 K had no further effect. Two distinct and simple models can account for this curious temperature dependence. By analyzing the kinetic and steady-state data, with reasonable estimates of the molar extinction coefficients of P(r) and P(g), we computed quantum efficiencies greater than 15% for the photoconversion at 300 K. We deduced that a conversion of "all P(r)" to "all P(g)" should produce a fractional absorbance change DeltaA/A at 620 nm equal to 0.1. If the chromatic adaptation response of intact F. diplosiphon shows the unusual temperature dependence reported here, the system P(r) right harpoon over left harpoon P(g) will be implicated in mediating this response.

Journal Article↗
PubMed1979

Comparison of the Molecular Weights of Proteins Synthesized by Isolated Chloroplasts with Those Which Appear during Greening in Zea mays.

The proteins of prolamellar bodies of etioplasts and of thylakoid membranes of greening and mature chloroplasts from Zea mays were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three classes of proteins were distinguished: those present in etioplasts and disappearing during greening, those absent in etioplasts and appearing during greening, and those present in both etioplasts and chloroplasts. The largest number of proteins belonged to this last class.The molecular weights of chloroplast thylakoid proteins were compared to the molecular weights of the membrane-associated proteins synthesized by isolated, mature chloroplasts. Thirteen of the 15 to 20 membrane-bound proteins made by isolated chloroplasts corresponded in size to proteins present in chloroplasts. Most of the 13 are present in both etioplasts and chloroplasts although a few were the same size as proteins which increase during greening. Production of most of the membrane proteins made in the plastids is not stringently regulated by light in vivo. The polypeptide subunits of the light-harvesting pigment-protein complex, the most abundant proteins of the chloroplast thylakoids, were absent from etioplasts. They were not synthesized by isolated chloroplasts.

Journal Article↗
PubMed1979

Comparison of Large Subunits of Type II DNA-dependent RNA Polymerases from Higher Plants.

Two-dimensional tryptic mapping of (125)I-labeled polypeptides has been employed to compare the large subunits of type II DNA-dependent RNA polymerases from maize, parsley (Petroselinum sativum), and wheat. Maps of the 220 kilodalton (kd) and 140 kd subunits from wheat RNA polymerase II differ from those of the corresponding subunits from parsley enzyme II. The 180 kd subunits from maize and parsley type II enzymes also yield dissimilar tryptic maps. Thus, despite similarities in molecular mass, the large subunits of wheat, parsley, and maize type II RNA polymerases are unique to each individual plant species.

Journal Article↗
PubMed1979

Control of Phycoerythrin Synthesis during Chromatic Adaptation.

Chromatic adaptation is the process by which blue-green algae alter the rates of biliprotein synthesis in response to changes in the color of available light. We have examined the control of phycoerythrin synthesis during the early stages of chromatic adaptation in Fremyella diplosiphon using fluorescence spectroscopy and (35)S-labeling of polypeptides. Phycoerythrin synthesis begins within 45 to 90 minutes after transfer of cells from red to green light, but is blocked by rifamycin. Transfer of cells from green to red light stops phycoerythrin synthesis with a t(1/2) = 45 minutes, as does the addition of rifamycin in green light. Transfer from green light to darkness slows but does not stop phycoerythrin synthesis. Gel electrophoresis of labeled polypeptides, both soluble and membrane-bound, shows that the synthesis of some polypeptides other than phycoerythrin are also affected by changes in light. These data suggest that chromatic adaptation involves gene regulation at the transcriptional level.

Journal Article↗
PubMed1979

Cyanobacterial ribonucleic acid polymerases recognize lambda promoters.

We compared the initiation specificities in vitro of deoxyribonucleic acid-dependent ribonucleic acid polymerases purified from two cyanobacteria, Fremyella diplosiphon and Anacystis nidulans, and from Escherichia coli. A restriction fragment made from lambda deoxyribonucleic acid was used as a template. The cyanobacterial and E. coli ribonucleic acid polymerases recognized the same lambda promoters but exhibited different sensitivities to the inhibitor heparin, suggsesting differences in the structure of the initiation complexes.

Bacteriophage lambda↗
PubMed1978

Changing postdoctoral career patterns for biomedical scientists.

Between 1973 and 1977 the total number of Ph.D.'s holding postdoctoral appointments in the biomedical sciences increased at a rate of more than 550 individuals (12.5 percent) per year. During this same period the total number of doctorates awarded each year in these disciplines showed very little change. The postdoctoral growth can be attributed to substantial increases in both the numbers of recent graduates taking postdoctorals and the length of stay on these appointments. The lack of alternative employment opportunities has contributed heavily to the postdoctoral buildup. Continued growth is likely to have important consequences for biomedical research and research training.

Biology↗
PubMed1978

Differential expression of the gene for the large subunit of ribulose bisphosphate carboxylase in maize leaf cell types.

Mesophyll cells and bundle sheath cells, the dimorphic photosynthetic cell types in the C4 plant Zea mays, differ in protein composition. In particular, the large subunit of the chloroplast enzyme ribulose-1,5-bisphosphate carbocylase (EC 4.1.1.39) is found entirely or almost exclusively in bundle sheath cells. The DNA sequence coding for this polypeptide is contained in the chloroplast DNA of both mesophyll cells and bundle sheath cells. RNA complementary to this coding sequence has been detected in RNA from bundle sheath cells, but is almost or entirely absent from mesophyll cells. Similarly, translatable mRNA for this polypeptide has been detected in RNA from bundle sheath cells, but not from mesophyll cells. Portions of the 4200 base pair maize plastid DNA sequence Bam 9 outside the large subunit gene region appear to be transcribed in mesophyll cells. Thus differential gene expression at the mRNA level has been directly demonstrated to occur in these two cell types.

Carboxy-Lyases↗
PubMed1978

Maize plastid gene expressed during photoregulated development.

RNAs larger than about 6 S prepared from etioplasts of dark-grown maize seedlings, and from plastids at later stages of light-induced development, were labeled in vitro and hybridized to separated fragments of maize chloroplast DNA digested with endonucleases. The major nonribosomal RNA present in developing plastids, but virtually undetectable in etioplasts, hybridizes to chloroplast DNA Bam fragment 8 and has been mapped on the maize plastid chromosome. Other aliquots of RNA from plastids were translated in a rabbit reticulocyte-derived system. Developing plastids, and mature chloroplasts, but not etioplasts, contain mRNA for an approximately 34,500 dalton polypeptide. The simultaneous appearance, during light-induced maize plastid development, of RNA which hybridizes to Bam 8 and is translated into a 34,500 dalton protein indicates that photoregulated expression of a single gene is being observed.

Journal Article↗
PubMed1978

Membrane proteins synthesized but not processed by isolated maize chloroplasts.

One-dimensional maps of proteolytic fragments generated by digestion with Staphylococcus aureus protease in sodium dodecyl sulfate (SDS) were used to identify three polypeptides synthesized by isolated Zea mays chloroplasts. This technique does not depend upon proper incorporation of the newly synthesized polypeptides into a more complex structure for their identification. The only preliminary purification required is electrophoretic separation on SDS-polyacrylamide gels. The pattern of radioactive fragments from labeled proteins which co-migrate with the alpha and beta subunits of chloroplast coupling factor (CF1) corresponds precisely to the pattern of stainable fragments derived from subunits of the purified enzyme. A 34,500-dalton protein is the major membrane-associated product of protein synthesis by isolated maize chloroplasts. From the similarity in the fragments formed by digestion with S. aureus protease, it appears that this radioactive protein is probably a precursor of a 32,000-dalton protein which is a component of the thylakoid. The alpha and beta subunits of CF1 newly synthesized by isolated chloroplasts are not fully extractable by procedures which normally solubilize the enzyme from membranes. The 34,500-dalton protein is not processed to the 32,000-dalton form in any great amount by isolated chloroplasts. A 19,000-dalton fragment of the 32,000-dalton protein is protected from digestion when thylakoids are treated with proteases, while the newly synthesized 34,500-dalton protein is fully susceptible. The isolated chloroplast does not appear to be able to fully integrate these newly made proteins into the membrane structure.

Cell Membrane↗
PubMed1978

Erythromycin resistance and the chloroplast ribosome in Chlamydomonas reinhardi.

Five classes of erythromycin-resistant mutants of Chlamydomonas reinhardi have been identified. Each class corresponds to a different genetic locus, three nuclear and two chloroplast. The three nuclear loci appear to be unlinked, while Conde et al. (1975) have shown that the two chloroplast loci are linked, but not allelic. Mutants in each class have a unique pattern of cross-resistance in vivo to other antibiotics (lincomycin, carbomycin, and cleocin) that affect chloroplast protein synthesis. The chloroplast ribosomes from each class have a distinctive erythromycin-binding reaction in vitro.--Haploid and diploid strains containing combinations of different genes affecting the chloroplast ribosome were constructed to probe ribosome structure. New phenotypes were obtained by such combinations, demonstrating interactions between the gene products of a number of loci specifying ribosome components.

Chlamydomonas↗