Search PubMed⌕ Search

Biomedical subjects

J Karn

Publications and source records attributed to J Karn.

At least 37 records · Page 2Linked to original sources
PubMed1989

Transformation of growth factor-dependent myeloid stem cells with retroviral vectors carrying c-myc.

Myeloid progenitor cells and macrophages derived from bone marrow and spleen were efficiently transformed in vitro by infection with Moloney-based retroviral vectors carrying a human c-myc gene. Infected cells were plated in agar in the presence of combinations of the murine lymphokines CSF-1, IL-3, GM-CSF and IL-1. Between 20% and 100% of the colony-forming cells in the initial bone marrow or spleen population could be infected and gave rise to drug-resistant colonies. A large fraction of the infected cells showed continued proliferation after transfer to liquid media and we have derived over 200 growth factor-dependent cell lines. These include adherent and non-adherent CSF-1 or GM-CSF dependent macrophages and macrophage precursors and cell lines which require complex combinations of growth factors for optimal growth. Each of the cell lines displays a unique pattern of expression of surface markers specific for the myeloid lineage including the Mac-1, Mac-2, Mac-3, Ser-4 and F4/80 antigens. Surface markers not specifically associated with the myeloid lineage such as the MHC class II antigens and the Fc-receptor; and surface markers normally associated with the B-cell and T-cell lineages such as B220, L3T4 and Thy1.2 are also found on these cell lines.

Animals↗
PubMed1989

Regulation of cell cycle duration by c-myc levels.

Early passage murine fibroblasts infected with retroviral vectors carrying human c-myc 'minigenes' express high levels of c-myc and have a dramatically shortened G1-phase of the cell cycle. Cells infected with viruses where c-myc is expressed from the viral LTR (MSN-4 virus) express more c-myc protein than cells infected with viruses where c-myc is expressed from the SV40 early promoter (NSM-7 virus). Populations of cells were infected with high titre viruses, selected for drug-resistance, pulse labelled with bromodeoxyuridine (BrdUrd) and chased in BrdUrd free media. This allows accurate, simultaneous, measurement of the rate of exit of unlabelled cells from G1 and progression of BrdUrd-labelled cells through S-phase. The length of the G1-phase in cell populations infected with the MSN-4 virus is 4.65 h, a reduction of nearly 30% compared to the G1-phase length of 6.50 h seen in cells infected with the VSN-2 control virus. Cells infected with NSM-7 virus show an intermediate phenotype and have a G1-phase of 5.25 h. The lengths of the S-phase (4.50 to 4.75 h) and G2 + M phases (2.75 h) were not significantly altered by exogenous c-myc expression. When chases are performed in growth-factor free media, the G1-phase of infected and non-infected cells is extended by approximately 2 h. Cells infected with the c-myc viruses continue to cycle more rapidly than uninfected cells. Growth factor-deprived cells, restimulated with serum, show similar alterations of the cell cycle kinetics. MSN-4 and NSM-7 infected cells, expressing high levels of c-myc, enter S-phase 2 to 4 h earlier, but less synchronously, than control cells, and sustain subsequent rounds of DNA synthesis, while control cells do not. However, cells carrying activated c-myc genes have nearly-normal morphologies and are not tumourgenic in syngenic mice. These results demonstrate that c-myc levels are rate limiting for events in G1, and the length of G1 varies proportionally with the level of exogenous c-myc expression.

Animals↗
PubMed1986

Immunological identification of the genes encoding the four myosin heavy chain isoforms of Caenorhabditis elegans.

The nematode Caenorhabditis elegans produces four distinct myosin heavy chain (MHC) isoforms, A, B, C, and D. The MHC A and MHC B proteins are coordinately expressed in the body wall muscle and are incorporated into different regions of a single kind of thick filament. MHC C and MHC D are exclusively produced in the pharyngeal muscle. Previous studies of mutations that affect MHC B have shown that this isoform is encoded by the unc-54 gene. Three other MHC genes, myo-1, myo-2, and myo-3, were isolated from a C. elegans genomic library by hybridization with fragments of the unc-54 gene. We have now established the MHC isoform encoded by each gene. Restriction fragments from each of these genes were cloned in the plasmid expression vector pUR288, producing fusion proteins between Escherichia coli beta-galactosidase and portions of the MHC rod domains of each gene. The hybrid proteins were screened with a panel of 18 isoform-specific monoclonal antibodies. The results demonstrate that myo-1 encodes MHC D, myo-2 encodes MHC C, and myo-3 encodes MHC A.

Animals↗
PubMed1985

Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans.

We have sequenced 11 representative mutations of the unc-54 myosin heavy chain gene of Caenorhabditis elegans that affect the synthesis, assembly or enzymatic activity of the encoded myosin heavy chain. Six of the sequenced unc-54 mutations cause premature termination of protein synthesis. Four mutations (e1092, e1115, e1213, e1328) were ochre mutations, one mutation (e903) was a frameshift, which caused premature termination at a nearby UGA terminator, and one mutation (e190) was a deletion that altered the reading frame and caused termination at an ochre codon. Two mutations (e675 and s291) were inphase deletions, which resulted in a shortened myosin rod segment. These aberrant myosins fail to assemble into normal thick filaments. The sequence alterations of the missense mutations (e1152, s74, s95) indicated amino acid residues that are critical for myosin function. The mutation e1152 causes the production of a myosin heavy chain that fails to assemble into thick filaments. It had two adjacent amino acid substitutions at the extreme amino terminus of the rod, indicating a role for subfragment-2 in thick filament assembly. Mutants homozygous for s74 or s95 are very slow-moving, although they make myosin heavy chains that assemble normally. The encoded amino acid substitutions of s95 and s74 are in the 23 X 10(3) Mr and 50 X 10(3) Mr domains of the myosin head, flanking the ATP binding site. The sequenced mutations are distributed throughout the gene in the order predicted from genetic fine-structure mapping experiments. Seven of eight point mutations isolated following ethylmethane sulphonate mutagenesis were G X C to A X T transitions. A single X-ray-induced allele proved to be a deletion of two adjacent thymidine residues. The three deletion mutations were found in a region of the myosin rod with numerous direct and inverted nucleotide sequence repeats, but their origin cannot be accounted for by homologous recombination. Instead, a comparison of the deletion junctions suggests that the deletions arose by a site-specific mechanism.

Animals↗
PubMed1984

A new selective phage cloning vector, lambda 2001, with sites for XbaI, BamHI, HindIII, EcoRI, SstI and XhoI.

An improved bacteriophage lambda cloning vector, lambda 2001, has been constructed. The phage includes a 34-bp polylinker oligonucleotide which introduces cleavage sites for XbaI, SstI, XhoI, EcoRI, HindIII and BamHI, and can accommodate 10-kb to 23-kb fragments. Inserts that destroy the BamHI or XhoI cloning sites may be recovered by excision at flanking sites in the polylinker sequence. Insertion of foreign DNA into lambda 2001 generates phage with a Spi- phenotype. The recombinant phage are able to grow on P2 lysogens but the parental vector phages are not. In the course of this work, the polylinker sequence was also introduced into M13mp8. This produced a new vector, M13mp12, with cloning sites for EcoRI, SmaI, XbaI, SstI, XhoI, BamHI, and HindIII.

Bacteriophage lambda↗
PubMed1984

Methods for cDNA cloning and sequencing tobacco mosaic virus RNA.

DNA complementary to tobacco mosaic virus (TMV) RNA (cDNA) was prepared by priming reverse transcription with synthetic oligonucleotides. The cDNAs terminated prematurely at many specific sites and no transcripts longer than about 2000 nucleotides were obtained. However, the entire 6395 nucleotide long TMV RNA could be copied into cDNA by specific priming with a series of 13 to 17 residue long oligonucleotides or by non-specific priming with short, 4 to 7 residue, oligonucleotides. A number of different priming methods were used to convert the cDNA into double-stranded DNA (dsDNA). The double-stranded cDNA was recovered by shotgun cloning into M13 and analysed by sequencing. The frequency at which cDNA clones were recovered has been used to compare various cDNA cloning strategies.

Base Sequence↗
PubMed1983

Periodic features in the amino acid sequence of nematode myosin rod.

Properties of the amino acid sequence of the nematode myosin rod region, deduced from cloned DNA, are analysed. The rod sequence of 1117 residues contains a regular region of 1094 residues, which has features typical of an alpha-helical coiled coil, followed by a short non-helical tailpiece at the carboxyl end. The hydrophobic amino acids show the expected seven-residue pattern a, b, c, d, e, f, g, which is modulated by a longer repeat of 28-residue zones. In addition, there are four one-residue insertions, or skip residues, at the ends of zones, at positions 351, 548, 745 and 970. Myosin is considerably less hydrophobic than tropomyosin or alpha-keratin and the outer surface of the coiled coil is covered by clusters of positive and negatively charged amino acid side-chains. Molecular models suggest that the coiled coil is continuous throughout the rod, with an approximately uniform left-handed twist, except for a few turns of helix near each skip region, where the twist flattens out to accommodate the extra residue. Fourier transforms of the amino acid profiles show strong periodicities based on repeats of seven residues (7/2 and 7/3) and 28 residues (especially 28/3 and 28/9). The positive and negative charges each have strong 28/3-residue periodicities that are out of phase with one another. The negative charges also show a 196/9-residue modulation frequency, which may reflect the presence of a 196-residue structural unit in muscle, approximately 2 X 143 A long. The distribution of charged amino acids suggests that electrostatic forces are dominant in forming the thick filament structure. Models that allow regular patterns of interacting charges are restricted and the simplest types are discussed.

Amino Acid Sequence↗
PubMed1983

The genes sup-7 X and sup-5 III of C. elegans suppress amber nonsense mutations via altered transfer RNA.

The sup-5 III and sup-7 X suppressors in C. elegans have previously been shown to have many genetic properties in common with tRNA nonsense suppressors of microorganisms. We report here the results of two lines of investigation into the molecular basis of these suppressors. In one, which sought to determine the nature of suppressible alleles, we demonstrate through DNA sequencing studies that a suppressible allele, unc-54(e 1300) I, of the myosin heavy chain gene contains a C leads to T substitution, which changes a glutamine codon to amber terminator at residue 1903. In the other approach, which sought to define the nature of the suppressing activity, we show through in vitro translation studies that tRNA fractions from the suppressor strains, but not wild-type, promote the specific readthrough of amber terminators of three different messenger RNAs. We conclude that sup-5 and sup-7 result in readthrough of amber terminators in vivo through an altered tRNA.

Alleles↗
PubMed1983

Protein structural domains in the Caenorhabditis elegans unc-54 myosin heavy chain gene are not separated by introns.

The 1,966-amino acid unc-54 myosin heavy chain sequence was determined from DNA sequence studies of the cloned gene. The gene is split by eight short introns, 48-561 base pairs long, and appears to lack a "TATA" box at its promoter. The physical map of the gene was aligned with the genetic map by locating two point mutations and three internal deletions: 0.01 map units correspond to approximately 5 kilobases. Comparison of the unc-54 protein sequence with the sequence of a second myosin heavy chain from nematode, indicates that the globular head sequence S-1 is more highly conserved than the alpha-helical coiled-coil rod. Major sites of proteolysis in S-1 are associated with variable sequences that have the characteristics of surface loops. In both genes there is no correlation between the positions of introns and the major protein structural domains.

Adenosine Triphosphate↗
PubMed1982

Periodic charge distributions in the myosin rod amino acid sequence match cross-bridge spacings in muscle.

The amino acid sequence of the rod portion of nematode myosin, deduced for the sequence of the unc-54 heavy chain gene of Caenorhabditis elegans, is highly repetitive and has the characteristics of an alpha-helical coiled coil. The molecular surface contains alternate clusters of positive and negative charge. Interactions between charge clusters on adjacent molecules could account for the observed spacing of the myosin cross-bridges in muscle. Calculations also suggest that the N-terminal third of the rod is only loosely associated with the thick filament backbone. Bending of the rod near the end of this region could allow the N-terminal section to act as a hinged arm during muscle contraction.

Amino Acid Sequence↗
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↗