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E M Tobin

Publications and source records attributed to E M Tobin.

48 records · Page 3Linked to original sources
PubMed1985

A chlorophyll a/b-protein encoded by a gene containing an intron with characteristics of a transposable element.

We have sequenced a genomic subclone (pLg AB19/H5c) of Lemna gibba nuclear DNA containing a complete chlorophyll a/b protein coding region and 5' and 3' flanking nucleotides. The coding region contains an intron of 84 nucleotides that has features characteristic of a transposable element. Evidence from S1 nuclease mapping experiments is consistent with correct transcription and splicing of the AB19 or another closely related intron-containing gene. The encoded precursor polypeptide of 264 amino acid residues has a predicted Mr of 28,327. Approximately 35 N-terminal residues are cleaved from this protein to form the mature apoprotein. We have used theoretical considerations of protein structure to propose an experimentally testable model of the structure of this protein in thylakoid membranes.

Amino Acid Sequence↗
PubMed1984

Demonstration of transcriptional regulation of specific genes by phytochrome action.

We have developed an in vitro transcription system that uses nuclei isolated from Lemna gibba G-3. The in vitro transcripts include sequences homologous to hybridization probes for the small subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], the light-harvesting chlorophyll a/b-protein, and rRNA. Light-harvesting chlorophyll a/b-protein sequences are transcribed to a greater extent in nuclei isolated from plants grown in darkness with 2 min of red light every 8 hr than in nuclei isolated from dark-treated plants. Furthermore, the amount of these transcripts measured in plants given a single minute of red light after dark treatment is increased over the amount measured in dark-treated plants. The effect of red light is at least partially reversible by 10 min of far-red light given immediately after the red light pulse. Transcription of both rRNA and small subunit sequences is also stimulated by a single minute of red light as compared to dark-treated tissue. However, the relative magnitudes of the increases compared to the dark levels are smaller than the increase seen for the chlorophyll a/b-protein, possibly because of the higher level of transcription of these sequences in the dark. The effect of red light on the transcription of small subunit and rRNA sequences is also reversible by immediate treatment with 10 min of far-red light. Pulse chase studies of dark-treated nuclei for up to 110 min do not show substantial turnover of in vitro labeled small subunit and chlorophyll a/b-protein transcripts. We therefore conclude that phytochrome action has induced specific changes in transcription of these genes.

Journal Article↗
PubMed1983

Nucleotide sequence encoding the precursor of the small subunit of ribulose 1,5-bisphosphate carboxylase from Lemna gibba L.G-3.

We have sequenced a cDNA clone, pLgSSU, which encodes the small subunit of ribulose 1,5-bisphosphate carboxylase of Lemna gibba L.G-3 a monocot plant. This clone contains a 832 basepair insert which encodes the entire 120 amino acids of the mature small subunit polypeptide (Mr = 14,127). In addition this clone encodes 53 amino acids of the amino terminal transit peptide of the precursor polypeptide and 242 nucleotides of the 3' non-coding region. Comparison of the nucleotide sequence of pLgSSU with Lemna gibba genomic sequences homologous to the 5' end of the cDNA clone suggests that nucleotides encoding four amino-terminal amino acids of the transit peptide are not included in the cDNA clone. The deduced amino acid sequence of the Lemna gibba mature small subunit polypeptide shows 70-75% homology to the reported sequences of other species. The transit peptide amino acid sequence shows less homology to other species. There is 50% homology to the reported soybean sequence and only 25% homology to the transit sequence of another monocot, wheat.

Amino Acid Sequence↗
PubMed1983

Nascent polypeptide chains exit the ribosome in the same relative position in both eucaryotes and procaryotes.

We located the polypeptide nascent chain as it leaves cytoplasmic ribosomes from the plant Lemna gibba by immune electron microscopy using antibodies against the small subunit of the enzyme ribulose-1,5-bisphosphate carboxylase. Similar studies with Escherichia coli ribosomes, using antibodies directed against the enzyme beta-galactosidase, show that the polypeptide nascent chain emerges in the same relative position in plants and bacteria. The eucaryotic ribosomal exit site is on the large subunit, approximately 75 A from the interface between subunits and nearly 160 A from the central protuberance, the presumed site for peptidyl transfer. This is the first functional site on both the eucaryotic and procaryotic ribosomes to be determined.

Escherichia coli↗
PubMed1983

Phytochrome Control of the Expression of Two Nuclear Genes Encoding Chloroplast Proteins in Lemna gibba L. G-3.

Hybridization probes for two nuclear-coded chloroplast proteins of Lemna gibba L. G-3 have been constructed in order to investigate phytochrome regulation of specific sequences. The first probe is a cDNA clone encoding the small subunit of ribulose 1,5-bisphosphate carboxylase. This probe was isolated from a set of Lemna cDNA clones in the bacterial plasmid pBR322. The second probe is a subclone of a genomic clone encoding the light-harvesting chlorophyll a/b-protein. This clone was isolated from a set of genomic clones constructed in the lambda vector Charon 4 with L. gibba DNA fragments generated by partial EcoR1 digestion. The identity of these clones was confirmed by in vitro translation of RNA which hybridized to the cloned DNA. Plants grown under continuous white light contain high concentrations of both RNA sequences; however, when these plants are put into darkness the concentration of these RNAs decreases rapidly relative to the total amount of RNA. Plants grown in the dark with intermittent red light (2 minutes/8 hours) and put into complete darkness for 8 days also contain lower concentrations of the sequences in the total RNA. One minute of red light after this dark period results in a rapid increase in the levels of RNA hybridizing to the probes. The effect of red light can be reversed by far-red light. These experiments demonstrate that phytochrome action can rapidly influence either the rates of transcription or the rates of degradation of these mRNAs.

Journal Article↗
PubMed1981

White Light Effects on the mRNA for the Light-Harvesting Chlorophyll a/b-Protein in Lemna gibba L. G-3.

Translation products of poly(A) mRNA isolated from Lemna gibba L. G-3 include a major polypeptide of 32,000 daltons which is immunoprecipitated by antiserum to chlorophyll a/b-protein from Chlamydomonas. This 32,000 dalton polypeptide represents a precursor to the light-harvesting chlorophyll a/b-protein of molecular weight 28,000 found in the thylakoid membranes of Lemna gibba. The amount of this translatable mRNA decreases relative to other translatable mRNAs when green plants grown in continuous white light are placed in darkness. This decrease occurs rapidly. The most rapid decline occurs during the first day; after 4 days of darkness, only a low level of this mRNA can be detected by in vitro translation. When the plants are returned to white light there is an increase in the relative level of this mRNA which can be easily detected within two hours. The in vivo synthesis of this protein has been assayed under the different light conditions. The light effects on the in vivo synthesis of the chlorophyll a/b-protein reflect the light effects on the translatable mRNA for the polypeptide. The results indicate that light induced changes in the synthesis, processing, or degradation of chlorophyll a/b-protein mRNA could account for the light-induced changes observed in the effective synthesis rates for the chlorophyll a/b-protein in vivo.

Journal Article↗
PubMed1980

Light Effects on the Synthesis of Ribulose-1,5-Bisphosphate Carboxylase in Lemna gibba L. G-3.

Placing light-grown Lemna gibba L. G-3 into the dark results in a changed pattern of protein synthesis. Although the amount of protein in the tissue and the over-all rate of incorporation of [(35)S]methionine into protein does not significantly decline during four days of darkness, the rate of synthesis of three polypeptides declines dramatically. One of these polypeptides is the chlorophyll a/b-binding protein and the two others are the large and small subunits of ribulose-1,5-bisphosphate carboxylase. The changed rates of synthesis of the two subunits were examined after transitions of plants from light to dark and dark to light. The in vivo synthesis of both subunits, while declining to a low level during four days of darkness, increases rapidly upon returning the plants to white light. In addition, the level of poly(A) mRNA coding for the precursor polypeptide of the small subunit of the enzyme falls to a low level in the dark and increases rapidly in response to white light. The increase in translatable mRNA for the small subunit is rapid enough to account for a major part of the increased synthesis of this subunit.

Journal Article↗
PubMed1978

Light regulation of specific mRNA species in Lemna gibba L. G-3.

Polyadenylated RNA was isolated from Lemna gibba L. G-3 and translated in a cell-free system from wheat germ. When plants were placed into complete darkness for 4 days, then returned to light for 18 hr, increased amounts of polyadenylated mRNA for at least two polypeptides were detected by in vitro translation over those amounts present in the dark. These two polypeptides have molecular weights of 32,000 and 20,000. The 20,000 dalton polypeptide was identified by immunoprecipitation as the precursor to the small subunit of the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39]. The polyadenylated mRNA that codes for the small subunit is not detectable by immunoprecipitation of translation products in the dark-treated tissue. Plants growing in the light actively synthesize both subunits of ribulose-1,5-bisphosphate carboxylase, but synthesis of these proteins was found to be greatly diminished in plants placed in darkness for 4 days. These results indicate that white light can dramatically affect the steady-state levels of specific polyadenylated mRNAs.

Journal Article↗
PubMed1975

Isolation and translation of plant messenger RNA.

A fraction of the RNA species isolated from Lemna gibba G-3 consists of molecules with attached sequences of polyadenylic acid. This polyadenylic acid-containing fraction, separated from total RNA by adsorption onto oligothymidylic acid-cellulose, was shown to be mRNA by its ability to serve as template in a cell-free translation system derived from wheat germ. The products of translation were characterized by electrophoresis. This method permitted the comparison of mRNA from plants grown under different light conditions. Such plants were shown to possess qualitative and quantitative differences in their mRNA complements.

Journal Article↗