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J Karn

Publications and source records attributed to J Karn.

68 records · Page 4Linked to original sources
PubMed1982

Sensitive detection of RNA using strand-specific M13 probes.

We have extended the method of Hu and Messing (Gene 17 (1982) 271-277) to prepare highly radioactive M13 probes suitable for use in RNA-DNA hybridization experiments. Single strands of M13 DNA carrying cloned sequences are rendered partially double-stranded by primed synthesis using a synthetic oligonucleotide primer complementary to a region 5' to the cloning site. The newly synthesized radioactive complementary strand is then covalently cross-linked to the M13 phage DNA by UV irradiation in the presence of 4,5,8-trimethylpsoralen (trioxsalen). Since the cross-linked probe is stable to heat denaturation, and the region of cloned sequence is kept single-stranded, these complexes may be used as strand-specific hybridization probes to detect RNA sequences under conditions which would denature DNA-DNA duplexes.

Base Sequence↗
PubMed1982

Phasmids: hybrids between ColE1 plasmids and E. coli bacteriophage lambda.

Plasmids carrying cloned lambda att sites may be integrated into the bacteriophage genome by the site-specific recombination mechanism of lambda. The cross, referred to as "lifting" the plasmid, requires mixed infection of an Escherichia coli strain carrying the plasmid with two appropriately constructed "lifting" lambda phages. One phage donates a short left arm and the other donates a short right arm. These two short arms are of insufficient length to produce a viable phage genome and yield no recombinants when crossed on standard bacteria. However, viable recombinants are obtained when the genome length is extended by integration of one or more plasmids. We call these recombinants phasmids. They contain multiple att sites introduced at the ends of the integrated plasmids, and in the presence of integrase, recombination between these att sites can be exploited to effect release of the plasmid components. These novel genetic elements can be used in a variety of ways as vectors in genetic manipulation experiments. Sequences cloned in phasmids may be studied as a component of either a plasmid and or of a phage, and easily interconverted between the two states.

Attachment Sites, Microbiological↗
PubMed1982

Nucleotide sequence of tobacco mosaic virus RNA.

Oligonucleotide primers have been used to generate a cDNA library covering the entire tobacco mosaic virus (TMV) RNA sequence. Analysis of these clones has enabled us to complete the viral RNA sequence and to study its variability within a viral population. The positive strand coding sequence starts 69 nucleotides from the 5' end with a reading frame for a protein of Mr 125,941 and terminates with UAG. Readthrough of this terminator would give rise to a protein of Mr 183,253. Overlapping the terminal five codons of this readthrough reading frame is a second reading frame coding for a protein of Mr 29,987. This gene terminates two nucleotides before the initiator codon of the coat protein gene. Potential signal sequences responsible for the capping and synthesis of the coat protein and Mr 29,987 protein mRNAs have been identified. Similar sequences within these reading frames may be used in the expression of sets of proteins that share COOH-terminal sequences.

Amino Acid Sequence↗
PubMed1980

Novel bacteriophage lambda cloning vector.

A simple method for generating phage collections representing eukaryotic genomes has been developed by using a novel bacteriophage lambda vector, lambda 1059. The phage is a BamHI substitution vector that accommodates DNA fragments 6-24 kilobases long. Production of recombinants in lambda 1059 requires deletion of the lambda red and gamma genes. The recombinants are therefore spi- and may be separated from the spi+ vector phages by plating on strains lysogenic for bacteriophage P2. Random fragments suitable for insertion into lambda 1059 are obtained by partial digestion of high molecular weight eukaryotic DNA with Sau3a. This restriction enzyme cleaves at the sequence G-A-T-C and leaves a 5'-tetranucleotide "sticky end." Because G-A-T-C extensions are also produced by BamHI cleavage, these fragments may be annealed directly to BamHI-cleaved lambda 1059. By using these methods, a set of clones covering the entire Caenorhabditis elegans genome was constructed. DNA segments which include the unc-54 myosin heavy chain gene have been isolated from this collection.

Animals↗
PubMed1977

Cyclic nucleotide phosphodiesterases of Hyalophora cecropia silkmoth fat body.

Two soluble forms of 3':5'-cyclic-nucleotide phosphodiesterase (o':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) were found in the larval fat body of the silkmoth Hyalophora cecropia. These differ in elution profile on Sephadex G-200, solubility in ammonium sulfate, metal ion requirements and kinetic properties. Phosphodiesterase I has Km values of 11 muM and 1.8 muM for cyclic AMP and cyclic GMP, respectively, has 5-fold greater maximal activity with cyclic AMP than with cyclic GMP, and is activated by Mg2+ and Co2+, and inhibited by EDTA. phosphodiesterase II has Km values of 625 muM and 125 muM for cyclic AMP and cyclic GMP, respectively, has similar maximal activity with both substrates, and is not activated by divalent metal ions or inhibited by EDTA. Cyclic nucleotides and methylxanthines competitively inhibit both enzymes. Phosphodiesterase is found in both soluble and particulate fractions of homogenates. Total activity is highest during the larval stage of the insect, drops markedly following pupation, and rises again during pharate adult development.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Selective effects of inhibitors of protein synthesis on metabolism of nuclear anc cytoplasmic proteins: evidence for coordinate synthesis of non-histone chromosomal proteins.

We have compared the effects of inhibitors of protein synthesis on the metabolism of nuclear and cytoplasmic proteins of HeLa S-3 cells. L-1-tosylamido-2-phenylethyl chloromethyl ketone, a potent inhibitor of polypeptide chain initiation, was shown to preferentially inhibit the synthesis of cytoplasmic proteins and of histones at concentrations that permit continued amino acid incorporation into nuclear non-histone proteins. Comparisons of the molecular weight distributions of newly synthesized proteins in the presence and absence of L-1-tosylamido-2-phenylethyl chloromethyl ketone have revealed striking differences between nuclear anc cytoplasmic protein fractions. Differential effects on the synthesis of cytoplasmic proteins, acid-soluble nuclear proteins, and residual nuclear proteins have also been obtained with the antibiotic, pactamycin, another inhibitor of polypeptide chain initiation. The incorporation of radioactive amino acids into nuclear non-histone proteins shows resistance to inhibition by pactamycin, but is strongly inhibited by agents such as puromycin and cycloheximide which block chain elongation. The possibility that proliferating cells have developed specialized mechanisms for the coordinate synthesis of chromosomal proteins, possibly involving polycistronic messenger RNAs, is tested and discussed.

Cell Nucleus↗