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Biomedical subjects

R G Herrmann

Publications and source records attributed to R G Herrmann.

At least 127 records · Page 7Linked to original sources
PubMed1983

Genes and transcripts for the polypeptides of the cytochrome b6/f complex from spinach thylakoid membranes.

Cytochrome b6/f complex was prepared from washed thylakoid membranes by a procedure involving detergent treatment and centrifugation in sucrose gradients. The complex is composed of at least four polypeptide species, cytochrome f which occurs in two variant forms (mol. wt. 34/33 kd), cytochrome b6 (23 kd), the high-potential Rieske iron-sulfur protein (19 kd) and a fourth subunit (17 kd) of unknown function. Transcripts for the cytochromes f, b6 and subunit 4 were found in plastid RNA, those for the Rieske iron-sulfur protein in cytosolic poly(A) RNA. Transcripts for cytochrome b6 and subunit 4 are translated in rabbit reticulocyte lysates into products of correct length. The Rieske iron-sulfur protein and the cytochrome f apoprotein appear to be made as precursors with excess sequences of 7 and 4 kd, respectively. Cytochrome f, cytochrome b6 and subunit 4 are encoded by uninterrupted plastid genes that are located in the large single-copy region of the circular DNA molecule. Each of these genes is present once per chromosome. Their location and direction of transcription have been determined by hybrid-selection mapping and by cell-free transcription/translation of various recombinant DNAs. The genes for cytochrome b6 and for subunit 4 lie near each other, but do not overlap. They are transcribed into a single message. The gene for cytochrome f maps 15 kbp away from this cluster, close to the 3' end of the gene for the large subunit of ribulosebisphosphate carboxylase/oxygenase, and is transcribed into a separate 4 kb long RNA. All these genes have the same polarities with respect to each other.

Journal Article↗
PubMed1983

Localization of the genes for the two chlorophyll a-conjugated polypeptides (mol. wt. 51 and 44 kd) of the photosystem II reaction center on the spinach plastid chromosome.

A core particle of the water-oxidizing photosystem II reaction center has been prepared from stacked spinach thylakoid membranes by a procedure involving extraction with the non-ionic detergent dodecyl-beta-D-maltoside and centrifugation in sucrose gradients. The protein-pigment complex consists of at least four polypeptide species: two components with mol. wts. of 51 and 44 kd which are conjugated with chlorophyll a and beta-carotene, the herbicide-binding protein of mol. wt. 32 kd and cytochrome b 559 (11 kd). The genes for the 51-and 44-kd polypeptides have been located on the circular 150-kbp spinach plastid chromosome. They were identified by hybrid-selection mapping, in vitro transcription-translation of recombinant DNAs and specific antisera which were used to characterize the translation products. The plastid chromosome carries one uninterrupted copy for each of these genes in its large single-copy region. The gene for the 51-kd protein (which probably bears the P(680) reaction center chlorophyll a) is located in close proximity to the gene for cytochrome b6, and some 70 kbp away from the gene for the ;32-kd' herbicide-binding protein of the reducing side of photosystem II. The gene for the 44-kd protein is situated halfway between these two genes adjacent to the gene for the P(700) chlorophyll a apoprotein of the photosystem I reaction center. Both photosystem II genes are transcribed into discrete RNA species in the same direction but from the opposite strand as the gene for the ;32-kd' protein.

Journal Article↗
PubMed1982

Immobilization of denatured DNA to macroporous supports: I. Efficiency of different coupling procedures.

Methods commonly used for covalent immobilization of single stranded DNA have been applied to several solid supports (Sephadex G-25 and Cellex 410) as well as to a number of macroporous materials (Sepharose C1-6B, C1-2B; Sephacryl S-500 and S-1000). Coupling efficiencies and stability of covalently bound DNA are compared for both classes of materials. The yields of the immobilization reaction for sonicated DNA are only 10-40% for G-25 and Cellex 410 in contrast to 60-80% for C1-6B and S-500. Under optimal conditions, up to 0.5 mg of DNA can be coupled initially per g of wet macroporous material. The immobilized DNAs are lost from the supports in a biphasic manner, with about 10-20% loss per day during the first 2-3 days at 45 degrees C, followed by only about 1% loss per day at the same temperature thereafter. The influence of the coupling procedure on the generation of mismatch effects has been studied in 2.4 M tetraethylammonium chloride solution for the hybrid formation between immobilized and mobile DNA. The degree of mismatch ranged from 0-3% and depended on the method of immobilization. The unspecific absorption of DNA on macroporous materials is sufficiently low to allow efficient hybrid selection. No size limitations have been observed when plastid mRNAs are selected by cloned fragments of plastid DNA immobilized to macroporous Sephacryl S-500.

Chromatography, Affinity↗
PubMed1980

Construction of a SalI/PstI restriction map of spinach chloroplast DNA using low-gelling-temperature-agarose electrophoresis.

The restriction endonucleases SalI and PstI cleave circular chloroplast DNA of spinach (Spinacia oleracea) into 12 and 10 fragments, respectively. The sum of the fragment sizes in each of the series is equivalent to the contour length of the molecule (about 95 Md). A physical map was constructed by sequential digestions using low-gelling-temperature agarose to avoid the necessity of extracting the fragments from the gel. The circular DNA molecule of spinach chloroplasts consists of two identical sequences (each about 15 Md) arranged as an inverted repeat separated by two single-copy regions of different sizes (about 52 and 13 Md).

Base Sequence↗
PubMed1980

The presence of DNA in ribosome-deficient plastids of heat-bleached rye leaves.

In leaves of rye seedlings (Secale cereale L.) grown at 32 degrees C the formation of plastid (70-S) ribosomes is specifically prevented. The resulting plastid-ribosome-deficient leaves can be used as a suitable system to identify chloroplast proteins which are translation products of cytosolic (80-S) ribosomes. The ribosome deficiency in plastids is accompanied by a bleaching of the leaves in light. In experiments aimed at finding the primary heat-sensitive event leading to ribosome deficiency the DNA of rye chloroplasts has been identified. Its properties are similar to those of chloroplast DNAs from other higher plants. The ribosome-deficient plastids isolated from heat-bleached rye leaves contained a DNA species which was indistinguishable from that of chloroplasts with regard to buoyant density in CsCl equilibrium gradients, reassociation properties and fragment patterns obtained upon cleavage by restriction endonucleases. Its quantity was comparable to that of chloroplast DNA of green leaves grown at a permissive temperature (22 degrees C). These results suggest that, unlike the effect in heat-bleached Euglena strains, lack of chloroplast DNA cannot be considered as the reason for the primary effect of high temperature in rye leaves but steps in the biosynthetic pathway of plastid ribosomes themselves must be affected more directly.

DNA↗
PubMed1979

Fractionation and identification of spinach chloroplast transfer RNAs and mapping of their genes on the restriction map of chloroplast DNA.

Spinach chloroplast 4S RNAs has been separated by two-dimensional polyacrylamide gel electrophoresis into about 35 species. After extraction from the gel, 27 of these RNA species were identified by aminoacylation as tRNAs specific for 16 amino acids. Individual tRNAs were labeled in vitro with 125I and hybridized to DNA fragments obtained by digestion of spinach chloroplast DNA with KpnI, PstI, SalI and XmaI restriction endonucleases. A minimum of 21 genes corresponding to tRNAs for 14 different amino acids have been localized on the restriction endonuclease cleavage site map of the DNA molecule. Of these, 15 genes corresponding to tRNAs for 12 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. Each copy of this repeat region contains a set of genes for the ribosomal RNAs and a gene for tRNA2Ile in the "spacer" sequence between the 16S and 23S ribosomal RNAs. The genes for tRNA1Ile, tRNA2Leu and tRNA3Leu also map in the repeat region, but outside the ribosomal DNA unit. At present, two more chloroplast tRNAs (for Pro and Lys) have been identified, but not mapped, while 4 unidentified 4S RNAs have been mapped in the large single-copy region of the DNA molecule. Evidence is presented that isoaccepting tRNA species can be transcripts from different loci.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Mapping of the ribosomal RNA genes on spinach chloroplast DNA.

Spinach chloroplast ribosomal RNAs have been hybridized to restriction endonuclease fragments of spinach chloroplast DNA. All three RNA species (23S, 16S and 5S) hybridized to a single large fragment when the DNA was digested with either Sall or Pstl. Hybridization of 23S RNA to fragments produced by Smal yielded two radioactive bands which corresponded to the bi-molar 2.5 X 10(6) and 1.15 X 10(6) Mr fragments. 16S RNA also hybridized to two, bi-molar Smal fragments (3.4 X 10(6) and 2.5 X 10(6) Mr) and 5S RNA hybridized to the 1.15 X 10(6) Mr bi-molar Smal fragment. The 23S RNA and 16S RNA cistrons were each also shown to contain a single EcoRI site. From the data it was possible to conclude that the ribosomal RNA genes are located on the inverted repeat region of the spinach chloroplast DNA restriction map [1,2], that the sequence of the cistrons is 16S - 23S - 5S and that the size of the spacer between the 16S and 23S RNA cistrons is approximately 0.90 X 10(6) Mr.

Chloroplasts↗
PubMed1975

Size, conformation and purity of chloroplast DNA of some higher plants.

1. Chloroplast DNA of Antirrhinum majus, Oenothera hookeri, Beta vulgaris and Spinacia oleracea band at the same buoyant density of 1.697 g-cm-3 in neutral CsCl equilibrium gradients. The corresponding nuclear DNAs band at 1.691, 1.703, 1.695 and 1.695 g-cm-3, respectively. The purity of chloroplast and nuclear DNA can be assessed objectively only in the cases of Antirrhinum and Oenothera. 2. Electron microscopic analysis of chloroplast DNA, purified in CsCl or CsCl/ethidium bromide gradients, revealed up to 80% circular molecules. Of these about 15% were of supertwisted conformation. Best yields of circular molecules were recovered when populations of unbroken chloroplasts were subjected to DNAase and phosphodiesterase treatment, and when the DNA was purified from viscous lysates by centrifugation into a CsCl cushion. Treatment of plastids with DNAase alone did not guarantee complete degradation of nuclear DNA. 3. The average contour length of the open circular chloroplast DNA molecules was basically similar for all four plants. They were 45.9 plus or minus 2.1 mum for Antirrhinum, 45.7 plus or minus 1.9 mum for Spinacia, 44.9 plus or minus 1.7 mum for Beta and 45.2 mum for Oenothera. This is comparable to the size derived for the coding capacity of chloroplast DNA from reassociation experiments. As much as 15% of the total population of circles in chloroplast DNA of Spinacia were circular dimers.

Centrifugation, Density Gradient↗