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Biomedical subjects

A Akrigg

Publications and source records attributed to A Akrigg.

16 recordsLinked to original sources
PubMed1992

Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector.

A defective adenovirus (Ad) type 5 E1- vector has been combined with the powerful constitutive cytomegalovirus (CMV) major immediate early (IE) promoter to produce a novel eukaryotic expression system. The Ad vector can replicate to high titres in 293 cells and then be used to infect a wide variety of non-permissive cell types. The Escherichia coli lacZ and CMV IE1 genes have been cloned to generate the Ad recombinants RAd35 and RAd31 respectively. In human fibroblasts infected with RAd35 beta-galactosidase (beta-gal) expression could be detected in virtually 100% of target cells, there was no detectable transcription from the Ad genome and extremely high levels of expression could be achieved with beta-gal representing the predominant cytoplasmic cellular protein. Additionally, a number of agents, including the CMV IE1 gene product (in RAd31) and forskolin, significantly enhanced expression from RAd35-infected human fibroblasts. Lower levels of constitutive beta-gal expression were obtained in RAd35-infected HeLa cells but again expression could be enhanced (up to 60 fold) by chemical inducing agents. Expression from the IE promoter in the Ad vector could be repressed by coinfection with CMV.

Adenoviridae↗
PubMed1991

HIV-1 indicator cell lines.

A simple quantitative bioassay for infectious HIV-1 has been developed. The assay is based on adherent CD4+ HeLa cell lines stably transfected with episomal vectors carrying the Escherichia coli beta-galactosidase gene under the control of the HIV long terminal repeat (LTR) promoter. HIV infection of these cell lines transactivates the LTR promoter inducing beta-galactosidase production. Infected cells and virus foci can be stained dark blue by the addition of the chromogenic substrate X-Gal. Alternatively, a readily automatable quantitative enzyme assay can be performed on the infected cultures. Because of its simplicity the bioassay may be useful for routine quantification of HIV-infected cultures, plaque purification, virus neutralization studies and for the screening of antiviral agents.

Biological Assay↗
PubMed1985

The structure of the major immediate early gene of human cytomegalovirus strain AD169.

The nucleotide sequence of the major immediate early (IE) gene of human cytomegalovirus strain AD169 was determined. The structure of the gene was examined by nuclease mapping and by sequence analysis of a cDNA clone made from IE mRNA. The gene encodes a spliced molecule of 1736 nucleotides, made up of four exon sequences of 121, 88, 185 and 1342 nucleotides. Three introns (827, 114 and 170n) were located near the 5' end of the gene. A single open reading frame starting in the second exon extends for 491 amino acids, corresponding to a protein of molecular weight 64000. The putative promoter region contains several short direct and inverted repeat sequences of 16, 18, 19 and 21 nucleotides, which extend 509n upstream from the transcription start site. The structure of the major IE gene and its protein product are discussed and compared with the corresponding IE gene from the Towne strain of HCMV.

Amino Acid Sequence↗
PubMed1984

Transcription of the immediate early genes of human cytomegalovirus strain AD169.

Cloned sub-genomic fragments of human cytomegalovirus strain AD169 were used to analyse immediate-early (IE) transcription in virus-infected cells. Transcriptionally active regions of the HCMV genome were identified by hybridising cytoplasmic IE poly(A)+-RNA with dot blots and Southern transfers of restriction endonuclease digests of recombinant plasmids. The size, number and, in some cases, the orientation of transcription of IE RNA species were determined. The most abundant IE mRNA (IE-1.95) was mapped at 0.0764-0.0865 map units. The transcription of two middle abundant (1.7 and 2.15 kb) IE RNAs was initiated immediately downstream, and in the same orientation as the IE-1.95 gene. A second transcriptionally active area was identified at 0.593-0.619 map units. Three mRNA species (IE-1.75, IE-3.8 and IE-4.8) were derived from this region. Additional minor IE transcription was also observed from other regions of the HCMV genome. Hybrid-selected translation was used to identify the polypeptides encoded by the major IE RNA species.

Base Sequence↗
PubMed1982

Use of recombinant plasmids to investigate the structure of the human cytomegalovirus genome.

Human cytomegalovirus (HCMV) DNA was digested with restriction endonucleases and the fragments characterized with respect to molecular weight and relative mole proportions. The terminal fragments were identified by digesting HCMV DNA with exonucleases before restriction endonuclease treatment and subsequent gel analysis. The HindIII fragments of HCMV DNA were cloned in Escherichia coli and recombinant plasmids were characterized by digestion with restriction endonucleases and by molecular hybridization with HindIII, Bg/II and XbaI fragments of the virus genome. Data from these experiments were used to construct physical maps of HCMV DNA for the HindIII, Bg/II and XbaI restriction endonucleases. The terminal regions of the genome and the region containing fragment HindIII M were shown to be heterogeneous.

Bacterial Proteins↗
PubMed1981

Molecular cloning of infectious DNA from human papovavirus BK in Escherichia coli.

Recombinant DNA constructed from unit length BK virus DNA and from several defective viral forms was cloned in Escherichia coli HB101. The cloned unit length BK virus DNA retained its infectivity for human embryonic kidney cells, whereas the cloned defective DNA showed no infectivity. Restriction endonuclease digestion of cloned defective DNA and purified virus DNA indicated that many of the defective forms contain re-iterated sequences.

Cloning, Molecular↗
PubMed1980

DNA gyrase stimulates transcription.

The nuclear DNA of HeLa cells can now be isolated unbroken and supercoiled. Using DNA gyrase and the untwisting enzyme, we have prepared an allomorphic series of templates derived from this nuclear DNA, and also from the circular DNA of the bacterial virus, PM2. We have then transcribed these templates using 2 different RNA polymerases--from wheat germ and Escherichia coli. Relaxed DNA is transcribed slowly by both polymerases. Supertwisting the naturally-supercoiled templates with gyrase slightly inhibits transcription by the bacterial polymerase but stimulates dramatically transcription by RNA polymerase II from wheat germ.

Animals↗
PubMed1979

Electron-microscopy of intact nuclear DNA from human cells.

Structures retaining many of the morphological features of nuclei may be released by lysing human cells in a non-ionic detergent and 2 M NaCl. Such nucleoids contain all the nuclear DNA packaged within a flexible cage of RNA and protein. HeLa nucleoids have been spread at an air-water interface and viewed in the electron microscope. A tangled network of superhelical fibres surrounds the collapsed cage. Irradiation with gamma-rays abolishes supercoiling and treatment with the untwisting enzyme or a low concentration of ethidium reduces it. A high concentration of ethidium induces supertwisting. The nuclear DNA of higher cells can be isolated naked, supercoiled and intact.

Cell Nucleus↗
PubMed1978

Extracellular manganese-stimulated deoxyribonuclease as a marker event in sporulation of Bacillus subtilis.

A considerable amount of Mn2+-stimulated DNAase (deoxyribonuclease) activity is released by Bacillus subtilis 168 during sporulation in a glucose-deficient medium; much smaller amounts are released during starvation for phosphate or nitrogen. Protein synthesis is required. Two forms of evidence are presented that production of the DNAase is associated with events late in stage II of sporulation. 19 Thymidine starvation, which inhibits the biochemical events associated with sporulation, also inhibits release of the DNAase. 2. Several asporogenous mutants blocked at stage II or earlier and unable to produce alkaline phosphatase (a stage-II event) do not produce the enzyme. Mutants blocked towards the end of stage II or later produce both enzymes. During sporulation of the wild-type strain, the DNAase appears about 1 h after alkaline phosphatase. The results suggest that production of the DNAase is controlled by a still-undiscovered stage-II genetic locus.

Bacillus subtilis↗
PubMed1978

Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis.

A DNAase (deoxyribonuclease) was isolated from culture supernatants of sporulating Bacillus subtilis 168. The purified enzyme migrated as a single band during polyacrylamide-gel electrophoresis. The enzyme differs from other DNAases of B. subtilis in molecular weight, metal-ion requirement and mode of action. The enzyme was inactive in the absence of metal ions, and exhibited optimum activity with 10 mM-Mn2+, although Mg2+, Cd2+ and Co2+ could also permit some activity. The pH optimum for the enzyme was pH 7.5, and it degraded linear-duplex DNA or closed-circular-duplex DNA to acid-soluble material. There was little or no activity on single-stranded DNA or rRNA. Sucrose-gradient analysis of the products of DNAase action on bacteriophage T7 DNA showed that endonucleolytic cleavage had occurred by the introduction of single-strand breaks in both strands of the duplex. The molecular weight of the enzyme was determined, by gel filtration on Sephadex G-75, to be 12000.

Bacillus subtilis↗
PubMed1970

Studies on the competence-inducing factor of Bacillus subtilis.

1. Aqueous extracts of competent cells of Bacillus subtilis 168I(-) were shown to contain a competence-inducing factor. The aqueous extracts were fractionated on DEAE-cellulose columns. 2. Those fractions from DEAE-cellulose columns containing the competence-inducing factor were shown to exhibit a powerful lytic effect on isolated cell walls of B. subtilis 168I(-). Little or no lytic activity was exhibited by the other fractions. 3. The kinetics of the lytic enzyme were investigated and found to be first-order. Treatment of cell wall lysates with 1-fluoro-2,4-dinitrobenzene suggested that the enzyme may be identical with N-acetylmuramyl-l-alanine amidase. The amino acid composition of the partially purified enzyme was determined. 4. It is suggested that competence-induction may be dependent on the limited action of the autolytic amidase.

Amino Acids↗