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L L Ku

Publications and source records attributed to L L Ku.

7 recordsLinked to original sources
PubMed1987

A novel method for the rapid detection of specific nucleotide sequences in crude biological samples without blotting or radioactivity; application to the analysis of hepatitis B virus in human serum.

The detection of a little as 0.2 pg (60,000 molecules) of hepatitis B viral (HBV) DNA in human serum samples in 4 h has been demonstrated using a solution-hybridization and bead-capture method. An amplification method based on chemically crosslinked oligodeoxyribonucleotides was coupled with a horseradish peroxidase-labeling scheme for the ultimate detection of the analyte. Two sets of HBV complementary synthetic oligodeoxyribonucleotide probes containing one of two types of single-stranded (ss) overhangs were employed. These ss overhangs were used to capture the probe-analyte complex onto a bead and subsequently to label it. Detection was achieved with either a chemiluminescent or colorimetric output substrate for the enzyme. Only in the presence of the virus was label specifically bound to the support. The assay was relatively unaffected by either sample composition or by the presence of heterologous nucleic acids.

Base Sequence↗
PubMed1987

High level expression of proinsulin in the yeast, Saccharomyces cerevisiae.

Human proinsulin (PI) has been expressed to a high level (100 mg/liter) as a human superoxide dismutase-PI fusion protein in the yeast, Saccharomyces cerevisiae. At the junction of the two proteins is a methionine residue, allowing PI to be released from the fusion by reaction with cyanogen bromide. The fusion is expressed using a regulated, hybrid promoter containing the regulatory region of the alcohol dehydrogenase II promoter and the 3' end of a glyceraldehyde-3-phosphate dehydrogenase promoter, allowing the recombinant yeast cells to be stably maintained. Production of the fusion protein is induced by growth in medium lacking a fermentable carbon source. The heterologous fusion protein is probably insoluble within the cell, since electron microscopy reveals the presence of 'inclusion bodies'. In a cell-free extract the fusion protein is also insoluble, but can be solubilized with sodium dodecyl sulfate, and cleaved with cyanogen bromide. The PI that is produced contains incorrect disulfide bonds. After sulfitolysis, the product can be easily purified, renatured, and processed to yield insulin.

Chromosome Mapping↗
PubMed1986

Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization.

Complementary DNA clones encoding the human kidney epidermal growth factor (EGF) precursor have been isolated and sequenced. They predict the sequence of a 1,207 amino acid protein which contains EGF flanked by polypeptide segments of 970 and 184 residues at its NH2- and COOH-termini, respectively. The structural organization of the human EGF precursor is similar to that previously described for the mouse protein and there is 66% identity between the two sequences. Transfection of COS-7 cells with the human EGF precursor cDNA linked to the SV40 early promoter indicate that it can be synthesized as a membrane protein with its NH2-terminus external to the cell surface. The human EGF precursor gene is approximately 110 kilobase pairs and has 24 exons. Its exon-intron organization revealed that various domains of the EGF precursor are encoded by individual exons. Moreover, 15 of the 24 exons encode protein segments that are homologous to sequences in other proteins. Exon duplication and shuffling appear to have played an important role in determining the present structure of this protein.

Amino Acid Sequence↗
PubMed1970

The ribosomes of pear fruit. Their synthesis during the climacteric and the age-related compensatory response to ionizing radiation.

Maceration at liquid nitrogen temperatures, use of poly-vinylpyrrolidone, and careful pH control are essential to the isolation of ribosomes and polysomes from deciduous fruit tissue. Characteristics of the ribosomes and constituent RNA are described. The distribution of ribosomes among monomer and polymer forms remains relatively constant until fruit reach the climacteric peak, after which there is a notable decline in the polymeric forms. In contrast to the relative stability in size distribution there is a marked change in ribosomal turnover during the climacteric rise. A transitory increase in rate of ribosomal synthesis is followed by a rapid decline coincident with the final portion of the climacteric rise. No incorporation of radioactive base into ribosomes could be detected once fruit had reached the climacteric peak.Coincidence of radioactivity with the ribosomal RNA on methylated albumin-Kieselguhr chromatograms and complete inhibition of ribosomal RNA synthesis by actinomycin D confirm that radioactive nucleotide was incorporated into newly synthesized ribosomes. Data are presented to distinguish between a cellular response to injury, as may result from the preparation of tissue slices, and the effects of physiological age. Superimposed intracellular radiation injury stimulates the synthesis of new ribosomes and underscores a major transition in the dynamics of the ribosomal system coincident with the climacteric rise.

Aging↗
PubMed1968

Cellular senescence, radiation damage to mitochondria, and the compensatory response in ripening pear fruits.

A compensatory response, viz. in vivo recovery from radiation damage to mitochondria, occurs in preclimacteric pear fruits (Pyrus communis L.) treated with ionizing radiation. The compensatory response is absent or markedly impaired in senescent fruits irradiated at or near the climacteric peak. Senescent cells failed to recover from harmful effects of radiation on: 1) mitochondrial yield, 2) in vivo incorporation of amino acids into mitochondrial protein, and 3) mitochondrial respiratory control and ADP/O. A diminished response to "split-dose" irradiation and a delayed rate of recovery confirmed the degeneracy and loss of compensatory power with cell age.A loss of restorative activity, especially in mitochondria that supply the cell with essential energy, may underlie the more obvious signs of cumulative stress that accompany cellular senescence. Use of ionizing radiation as an investigative tool and the molecular implications of radiation damage, recovery, and cellular senescence are discussed.

Journal Article↗