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J Y Sheen

Publications and source records attributed to J Y Sheen.

8 recordsLinked to original sources
PubMed1988

A simple phase-extraction assay for chloramphenicol acyltransferase activity.

A simple and convenient phase extraction assay for chloramphenicol (Cm) acetyltransferase (CAT) activity has been developed, based on the enzymatic butyrylation of radiolabelled Cm. The assay is linear over two to three orders of magnitude of enzyme concentration, is highly sensitive, and substantially less expensive than all presently available alternatives. Methods for convenient CAT assay, adapted for mammalian cells, plant protoplasts, and mammalian cell culture supernatants, are described. These methods should also simplify measurement of CAT activity in other organisms, such as yeast and bacteria. In addition, a simple pre-extraction procedure is presented for purifying radiolabelled Cm which allows a 25-fold increase in sensitivity using tritiated substrates.

Animals↗
PubMed1988

Differential Expression in Bundle Sheath and Mesophyll Cells of Maize of Genes for Photosystem II Components Encoded by the Plastid Genome.

We have explored the cell-specific expression of plastid genes encoding five photosystem II proteins (PSII-A, -B, -C, -D and cytochrome (Cyt) b-559 encoded by plastid genes psbA, B, C, D and E plus F, respectively) and one encoding the photosystem I protein PSI-A1 in bundle sheath and mesophyll cells (BSC and MC) at different stages of photoregulated development in leaves of dark-grown maize seedlings. Two types of cell-specific mRNA ccumulation patterns have been discerned. First is the PSII-A type: the mRNA is present in small but about equal amounts in both cell types in etiolated leaves; then, upon illumination, the level of mRNA increases progressively and remains high in MC but not BSC of green leaves. Second is the Cyt b-559 type: MC of etiolated leaves have about twice as much of this mRNA as do BSC; about a twofold to threefold transient increase is induced by light in MC but not in BSC. The genes for the PSII-C and -D proteins are complementary to a sizable family of transcripts over a large range of sizes; the patterns of changes in pools of some of these transcripts are of the PSII-A type and others are of the Cyt b-559 type. The DNA region encoding the PSII-B protein also hybridizes to multiple transcripts; among them is a prominent 2.2 kilobase transcript that follows the Cyt b-559 accumulation pattern. In contrast, the pattern of PSI-A1 mRNA accumulation is the same in both cell types. Accumulation of the PSII-B and -D proteins is light-dependent and coordinate. Both are much more abundant in MC than in BSC. PSII-A is detectable in MC of etiolated leaves and increases during photoregulated chloroplast maturation in both cell types, however, to a much greater extent in MC. We conclude that the expression of each of these plastidencoded photosystem II genes in BSC and MC is photoregulated differently; both transcript abundance and translation are regulated.

Journal Article↗
PubMed1987

Differential expression of C4 pathway genes in mesophyll and bundle sheath cells of greening maize leaves.

Pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase, and NADP-malate dehydrogenase function in a series of reactions for fixing CO2 in mesophyll cells and NADP-malic enzyme (ME) catalyzes the production of CO2 and NADPH in bundle sheath cells of maize which is a NADP-ME type C4 plant. Northern blot analyses with cDNA clones for pyruvate orthophosphate dikinase and phosphoenolpyruvate carboxylase and in vitro translation-immunoprecipitation experiments with antiserum to NADP-malate dehydrogenase showed that pools of transcripts of these three genes grow and shrink coordinately in mesophyll cells but not in bundle sheath cells upon illumination of dark-grown maize seedlings. Western blot analyses indicated that the protein levels of phosphoenolpyruvate carboxylase and pyruvate orthophosphate dikinase are low in dark-grown maize seedlings and increase progressively following light-induced transient accumulation of their mRNAs in mesophyll cells. These proteins continue to accumulate and plateau in late-greening and green leaves in spite of a rapid drop in the sizes of their mRNA pools. Surprisingly, relatively large amounts of NADP-malate dehydrogenase are present in mesophyll cells of etiolated leaves despite the low level of the corresponding mRNA. No phosphoenolpyruvate carboxylase or NADP-malate dehydrogenase were detected in bundle sheath cells. On the other hand, the ME gene responds to light induction at both the transcriptional and translational levels only in bundle sheath cells. Moreover, the steady-state level of ME mRNA stays high in late-greening and green leaves in contrast to the rapid decline of mRNA levels of three other C4 pathway genes in mesophyll cells. In addition, low levels of both the mRNA and protein encoded by the PPDK gene were detected in bundle sheath cells. These levels were not influenced by light as distinguished from the patterns observed in mesophyll cells.

Centrifugation, Density Gradient↗
PubMed1986

Expression of the ribulose-1,5-bisphosphate carboxylase large subunit gene and three small subunit genes in two cell types of maize leaves.

Transcripts of three distinct ribulose-1,5-bisphosphate carboxylase (RuBPC) small subunit (SS) genes account for approximately 90% of the mRNA for this protein in maize leaves. Transcripts of two of them constitute >80% of the SS mRNA in 24-h greening maize leaves. The third gene contribute approximately 10%. Transcripts of all three nuclear-encoded SS genes are detectable in bundle sheath (BSC) and mesophyll cells (MC) of etiolated maize leaves. The level of mRNA for each gene is different in etioplasts of MC but all drop during photoregulated development of chloroplasts in MC and follow a pattern of transitory rise and fall in BSC. The amounts of LS and SS proteins continue to increase steadily well after the mRNA levels reach their peaks in BSC. The molar ratio of mRNA for chloroplast-encoded RuBPC large subunit (LS) to the nuclear genome encoded SS is about 10:1 although LS and SS proteins are present in about equimolar amounts.

Journal Article↗
PubMed1986

Differential expression of six light-harvesting chlorophyll a/b binding protein genes in maize leaf cell types.

Bundle sheath chloroplasts of maize leaves contain about one-fourth as much light-harvesting chlorophyll a/b binding protein of photosystem II (LHCP-II) as do mesophyll chloroplasts. We have determined that this difference is, in part, the result of differential expression of different LHCP-II genes. We have prepared and partially characterized cDNA clones specific for six LHCP-II genes of maize. Transcripts of these six LHCP-II genes are present at vastly different levels and account for about 95% of total LHCP-II mRNAs in bundle sheath and mesophyll cells of illuminated dark-grown maize leaves. Three genes are preferentially expressed in mesophyll cells, and their mRNAs constitute about 54% of the total LHCP-II transcripts in greening (24 hr) maize leaves. Two genes are expressed equally in bundle sheath and mesophyll cells. Most interestingly, the RNA of one gene that contributes about 8% of the total LHCP-II transcripts in leaves greening for 24 hr is present at a much higher level in bundle sheath than in mesophyll cells. Moreover, immunoblot analysis of maize thylakoids reveals at least five sizes of LHCP-II; these also differ from one another in their relative abundance in bundle sheath and mesophyll cells of developing maize leaves.

Chlorophyll↗
PubMed1985

Differential expression of the ribulose bisphosphate carboxylase large subunit gene in bundle sheath and mesophyll cells of developing maize leaves is influenced by light.

We have investigated the influence of light on the mRNA of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in mesophyll and bundle sheath cells of the C(4) plant Zea mays. The gene is transcribed in both cell types in leaves of seedlings grown in the dark. However, upon illumination, the level of mRNA declines and becomes undetectable in mesophyll cells after 72 hours. On the contrary, in bundle sheath cells the two transcripts of the same gene increase to peak after 24 hours of greening and then decrease to a steady state level. This study was made possible by the development of improved methods for the isolation of two maize leaf cell types suitable for the extraction of high quality RNAs at different developmental states, i.e. etiolated, greening and green.

Journal Article↗
PubMed1983

Unequal crossing-over associated with asymmetrical synapsis between nomadic elements in the Drosophila melanogaster genome.

The molecular structure of reciprocal duplications and deficiencies produced by unequal crossing-over at the white (w) locus of Drosophila melanogaster females heterozygous for the alleles w(a) and w(a4) has been examined. A transposable, copia-like element is found at the rearrangement breakpoints. Further characterization indicates that asymmetrical pairing between two copies of this element, which are at least 60 kilobases apart in the parental chromosomes, followed by a crossover within the paired elements, is responsible for the duplication and deficiencies observed. The frequency of these events is high compared with normal homologous exchange, implying that synaptic pairing during meiosis must be sufficiently flexible as to allow efficient recognition of sequences located in nonidentical positions on homologous chromosomes. These results suggest a possible mechanism for the generation of tandem duplications in eukaryotic organisms.

Journal Article↗