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C J Leaver

Publications and source records attributed to C J Leaver.

At least 73 records · Page 4Linked to original sources
PubMed1983

The alpha-subunit of the maize F(1)-ATPase is synthesised in the mitochondrion.

The F(1)-ATPase complex has been purified from maize (Zea mays L.) mitochondria and shown to consist of five subunits with mol. wts. of 58 000 (alpha), 56 000 (beta), 35 000 (gamma), 22 000 (delta) and 8000 (epsilon). The alpha-subunit co-migrates on one- and two- dimensional isoelectric focussing-SDS polyacrylamide gels with the major polypeptide synthesised by isolated mitochondria. One-dimensional proteolytic peptide mapping and immunoprecipitation confirms that the alpha-subunit is a mitochondrial translation product and therefore presumably encoded in mitochondrial DNA. This contrasts with the situation in animal and fungal cells where all five subunits of the F(1)-ATPase are encoded by the nuclear genome and synthesised on cytosolic ribosomes.

Journal Article↗
PubMed1981

The Zea mays mitochondrial gene coding cytochrome oxidase subunit II has an intervening sequence and does not contain TGA codons.

Cross hybridization between maize mitochondrial DNA fragments and a specific yeast mitochondrial DNA probe from the oxi 1 gene has been used to identify and isolate the maize mitochondrial gene coding cytochrome oxidase subunit II, mox 1. The DNA sequence reveals two coding regions separated from each other by a single centrally located intervening sequence. Hybridization of mox 1 DNA probes to mitochondrial RNA from plants shows that te gene is transcribed and indicates that several transcripts are spliced. TGA codons, which code Trp in the mitochondria of all species examined to date, do not occur in this gene. However, alignment of the mox 1 gene sequence with the amino acid sequences of subunit II from other organisms strongly suggests that codon CGG (normally Arg) codes for Trp in maize mitochondria, in addition to the standard Trp codon TGG.

Base Sequence↗
PubMed1981

Synthesis of Chloroplast Proteins during Germination and Early Development of Cucumber.

Cell-free protein synthesizing systems have been used to study the developmental changes in the synthesis of chloroplast proteins in the cotyledons of cucumber seedlings grown in the light or in the dark. Escherichia coli and wheat germ in vitro protein synthesizing systems have been used to assay the changes in the levels of the mRNA's coding for ribulose 1,5-bisphosphate carboxylase (RuBPCase). The large subunit of cucumber RuBPCase has been identified among the translation products of the E. coli system. The wheat germ system translates the cucumber mRNA coding for the small subunit of RuBPCase to produce a 25,000 molecular weight precursor polypeptide. Plastids isolated from light-grown cotyledons were used to study developmental changes in their capacity to synthesize protein. The data obtained indicate that in the light there is an initial 48-hour period of accumulation of the mRNA's coding for the large and small subunits of RuBPCase, coupled with an increase in the capacity of the isolated plastids to synthesize protein. This is followed by a decline. This decline is not reflected in the accumulation of RuBPCase in the cotyledons which remains constant over the period of study.

Journal Article↗
PubMed1980

Nuclear and cytoplasmic genes controlling synthesis of variant mitochondrial polypeptides in male-sterile maize.

The polypeptides synthesized in vitro by mitochondria isolated from etiolated maize shoots of a number of different nuclear and cytoplasmic genotypes have been compared by using polyacrylamide gel electrophoresis. We have previously shown that mitochondria from maize plants carrying the T or C forms of cytoplasmically inherited male sterility (cms-T and cms-C mitochondria) can be distinguished from each other and from the mitochondria of normal (N) plants by the synthesis of a single additional or variant polypeptide species. Using lines that carry the T cytoplasm, and that differ principally in the presence or absence of nuclear "restorer" alleles that suppress the male-sterile phenotype, we find that these nuclear genes specifically suppress synthesis of the 13,000 M(r) variant polypeptide. A 21,000 M(r) polypeptide that is synthesized by N mitochondria is not detectable among the translation products of cms-T mitochondria from either restored or nonrestored lines. Results obtained with a number of lines possessing dominant restorer alleles from different sources indicate that it is the restorer gene at the Rf(1) locus that is primarily responsible for regulating synthesis of the 13,000 M(r) polypeptide. Mitochondria from lines with the S form of cytoplasmic male sterility (cms-S) were found to synthesize a group of minor polypeptides, ranging in molecular weight from 42,000 to 88,000, which were not detected in N, cms-T, or cms-C mitochondria. In the case of the S and C forms of male sterility no differences were found between the translation products of mitochondria from restored and nonrestored lines.

Journal Article↗
PubMed1980

Regulation of Glyoxysomal Enzymes during Germination of Cucumber: 3. IN VITRO TRANSLATION AND CHARACTERIZATION OF FOUR GLYOXYSOMAL ENZYMES.

Monospecific antibodies raised against four glyoxysomal enzymes (isocitrate lyase, catalase, malate synthase, and malate dehydrogenase) have been used to detect these proteins among the products of in vitro translation in a wheat germ system programmed with cotyledonary RNA from cucumber seedlings. In vitro immunoprecipitates were compared electrophoretically with the same enzymes labeled in vivo and also with the purified proteins. Isocitrate lyase yields two bands on sodium dodecyl sulfate-polyacrylamide gels, as synthesized both in vitro (61.5K and 60K products) and in vivo (63K and 61.5K polypeptides). Both the 63K and 61.5K subunits can also be demonstrated for the isolated enzyme. The two subunits are antigenically cross-reactive and yield similar electrophoretic profiles upon partial proteolytic digestion. A larger subunit is seen in vitro than in vivo for both malate dehydrogenase (38K versus 33K) and catalase (55K versus 54K); this suggests a need for processing which is often a characteristic of proteins that must be transported across or into membranes. Malate synthase has a molecular weight of 57K both in vitro and in vivo, but the isolated enzyme is a glycoprotein, containing N-acetyl glucosamine, mannose, and possibly also fucose and xylose. This indicates that the polypeptide portion of the isolated enzyme is smaller than the in vitro product and suggests processing of malate synthase also. None of the other three enzymes appears to be glycosylated. The implications of these size differences for the compartmentalization of matrix and membrane-bound glyoxysomal enzymes are discussed.

Journal Article↗
PubMed1980

Regulation of glyoxysomal enzymes during germination of cucumber. Temporal changes in translatable mRNAs for isocitrate lyase and malate synthase.

The relative levels of translatable messenger RNA for isocitrate lyase and malate synthase were determined in the dry seed and for the first seven days of development of cucumber cotyledons. After extraction and quantification of total and poly(A)-rich RNA each day, the RNA fractions were translated in an optimized wheat germ system and the specific polypeptides were immunoprecipitated quantitatively. The radiolabeled isocitrate lyase and malate synthase polypeptides were then fractionated on dodecylsulphate/polyacrylamide gels, visualized by exposure to X-ray film and quantified densitometrically. The relative levels of translatable messenger RNA for these enzymes rise and fall with a developmental program similar to the enzyme activities, but preceding the latter by about one day. This implies that the rise in enzyme activity is dependent upon a prior postgerminative increase in translatable messenger RNA for the enzymes. These studies also suggest that messenger RNA levels may be regulated, at least in part, by light.

Glyoxylates↗
PubMed1979

In Vitro Study of Mitochondrial Protein Synthesis during Mitochondrial Biogenesis in Excised Plant Storage Tissue.

Mitochondrial biogenesis was induced in Jerusalem artichoke (Helianthus tuberosus) tuber by aging tissue discs in distilled water for up to 26 hours. Changes in the purified mitochondrial fraction during aging included an increase in both protein content and specific respiratory activity. Using intact isolated mitochondria, conditions were optimized for incorporation of radioactive amino acid into protein. Incorporation was dependent upon the supply of an oxidizable substrate or an external ATP-generating system and showed characteristic sensitivity to inhibitors of protein synthesis. Aging of the tissue resulted in a 3-fold increase in the rate of in vitro incorporation of [(35)S]methionine into mitochondrial protein. An analysis of the free amino acid pool in the mitochondrial fraction showed that the decrease in methionine level during aging of intact tissue was sufficient to account for the increased rate of protein labeling. The activation of mitochondrial biogenesis which occurs after slicing is not dependent on an increase in the capacity of mitochondria to synthesize protein as assayed in vitro.Analysis, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, showed that isolated mitochondria synthesized about 15 polypeptides in the molecular weight range 8,000 to 55,000. As aging proceeded, significant changes were observed in the relative rates of labeling of three out of the eight major polypeptides synthesized by mitochondria in vitro.

Journal Article↗
PubMed1978

Low-molecular-weight (4.5S) ribonucleic acid in higher-plant chloroplast ribosomes.

A species of RNA that migrates on 10% (w/v) polyacrylamide gels between 5S and 4S RNA was detected in spinach chloroplasts. This RNA (referred to as 4.5 S RNA) was present in amounts equimolar to the 5S RNA and its molecular weight was estimated to be approx. 33 000. Fractionation of the chloroplast components showed that the 4.5S RNA was associated with the 50 S ribosomal subunit and that it could be removed by washing the ribosomes with a buffer containing 0.01 M-EDTA and 0.5 M-KCl. It did not appear to be a cleavage product of the labile 23 S RNA of spinach chloroplast ribosomes. When 125I-labelled 4.5 S RNA was hybridized to fragments of spinach chloroplast DNA produced by SmaI restriction endonuclease, a single fragment (mol.wt. 1.15 times 10(6)) became labelled. The same DNA fragment also hybridized to chloroplast 5 S RNA and part of the 23 S RNA. It was concluded that the coding sequence for 4.5 S RNA was part of, or immediately adjacent to, the rRNA-gene region in chloroplast DNA . A comparable RNA species was observed in chloroplasts of tobacco and pea leaves.

Chloroplasts↗
PubMed1978

Variation in mitochondrial translation products associated with male-sterile cytoplasms in maize.

Mitochondria were isolated under sterile conditions from maize lines with normal cytoplasm and with T, C, and S sources of cytoplasmic male sterility. Protein synthesis by these mitochondria was dependent upon the presence of ADP and an oxidizable substrate or an energy generating system and displayed characteristic sensitivity towards inhibitors of protein synthesis. Approximately 20 polypeptides were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis of the [(35)S]methionine-labeled mitochondrial translation products. Mitochondria from the T and C cytoplasms were characterized by the synthesis of a single additional or variant polypeptide.These observations are discussed with reference to the possible mechanisms of male sterility and the susceptibility of plants with T cytoplasm to Helminthosporium maydis, race T. Our results also suggest a potentially rapid method of classifying sources of cytoplasmic male sterility that could be used in conjunction with existing methods, which depend on patterns of fertility restoration after an extended breeding program.

Journal Article↗
PubMed1978

Regulation of Glyoxysomal Enzymes during Germination of Cucumber: I. Developmental Changes in Cotyledonary Protein, RNA, and Enzyme Activities during Germination.

Developmental patterns of glyoxylate cycle and photosynthetic activities have been correlated with electrophoretic profiles of cotyledonary RNA and protein in both light- and dark-grown cucumber seedlings (Cucumis sativus L.) Cytoplasmic rRNA increases 10-fold between days 0 and 5, and the steepest increase coincides with the most rapid rise in activities of the glyoxysomal enzymes, isocitrate lyase and malate synthase. Chloroplast rRNA and ribulose bisphosphate (RuBP) carboxylase begin rising at day 3, followed about a day later by increases in glyoxylate reductase activity and chlorophyll content. Of these phototrophic indicators, only chlorophyll requires light for its initial appearance. Sodium dodecyl sulfate gel electrophoresis of total and soluble cotyledonary protein showed several developmental patterns, including: (a) progressive disappearance of storage protein present initially in particulate form; (b appearance and subsequent disappearance of a family of polypeptides identified by molecular weight, developmental profile, and density gradient centrifugation as subunits of glyoxysomal enzymes; and (c) appearance and progressive increase (in both light- and dark-grown cotyledons) of the large and small subunits of RuBP carboxylase, as well as other polypeptides presumably of chloroplast and peroxisomal origin.

Journal Article↗
PubMed1976

Higher-plant mitochondrial ribosomes contain a 5S ribosomal ribonucleic acid component.

Ribosomes from higher-plant mitochondria contain 5S rRNA, in contrast with the mitochondrial ribosomes of animals and fungi, in which such a component has not been detected. In common with the ribosomes of prokaryotes and chloroplasts, higher-plant mitochondrial ribosomes do not appear to contain an RNA equivalent to the 5.8 S rRNA that is found in eukaryoytes hydrogen-bonded to the largest of the cytoplasmic rRNA species.

Electrophoresis, Polyacrylamide Gel↗
PubMed1974

Caution in the interpretation of plant ribosome studies.

Published methods of ribosome isolation from plant tissues result in a partial and selective recovery of the total ribosome population. The enrichment of polyribosomes obtained may give a false impression of the change in translational capacity of the tissue under study.

Cell Fractionation↗
PubMed1973

Molecular integrity of chloroplast ribosomal ribonucleic acid.

The majority of chloroplast 1.1x10(6)-mol.wt. rRNA molecules are nicked at specific points in the polynucleotide chain, the molecules being kept intact at low temperatures by their secondary structure. Conditions that break hydrogen bonds and lead to loss of secondary structure cause dissociation of the molecule.

Chloroplasts↗
PubMed1972

Characterization of Short Time Labeled Adenosine Monophosphate-rich Ribonucleic Acids of Soybean.

The total population of newly synthesized (32)P-AMP-rich RNA has been separated into two major types based on repeated fractionation on methylated albumin-kieselguhr columns. The purified D-RNA which elutes, under our experimental conditions, primarily in the salt gradient has a GMP/AMP ratio of about 0.8 and an AMP + UMP content of about 56 mole per cent. The purified TB-RNA which preferentially remains bound to the column in the salt gradient has a GMP/AMP ratio of about 0.4 to 0.45 and an AMP + UMP content of about 65 mole per cent. In addition to being distinguished by their fractionation on the methylated albumin-kieselguhr column and base composition analysis, purified D-RNA and TB-RNA have different size distributions on sucrose gradient and acrylamide gel fractionation, are differentially associated with polyribosomes and have different stabilities in the tissue.

Journal Article↗