Sequence analysis of two HTLV type I infections imported to Germany.
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DNA, Viral↗
Biomedical subjects
C Kücherer
Publications and source records attributed to C Kücherer.
Association of human immunodeficiency virus Nef protein with actin is myristoylation dependent and influences its subcellular localization.
Human immunodeficiency virus (HIV) Nef functions are thought to be mediated via interactions with cellular proteins. Utilizing zone velocity sedimentation in glycerol gradients we found that recombinant HIV-1 Nef non-covalently associates with actin forming a high-molecular-mass complex of 150-300 kDa. This Nef/actin complex was present in human B and T lymphocytes but not in insect cells and was dependent on the N-terminal myristoylation of Nef, whereas the SH3-binding proline motif of Nef was not involved. Despite being myristoylated, HIV-2 Nef did not associate with actin. This might reflect differences in the subcellular localization of Nef since cell-fractionation experiments revealed that HIV-1 Nef was virtually exclusively localized in the cytoskeletal (detergent-insoluble) fraction whereas HIV-2 Nef had significantly reduced affinity for the cytoskeleton. Colocalization experiments in HIV-1-infected CD4+ fibroblasts revealed that Nef/actin complexes may also exist in HIV-infected cells. This novel interaction of HIV-1 Nef with actin provides insight into the association of Nef with cellular structures and reveals general differences in the interactions of the Nef proteins from HIV-1 and HIV-2.
Actins↗
A multicentre quality assessment study to monitor the performance of HIV-1 PCR.
Eleven German laboratories and one Swiss laboratory initiated a quality assessment study to evaluate the specificity and sensitivity of their polymerase chain reaction (PCR) for detection of HIV-1 DNA. Following its own PCR protocols, each laboratory tested a panel of ten coded samples consisting of cell pellets containing 0, 0.1, 1, 10, 10(2), 10(3) and 10(4) ACH-2 cells per 1.5 x 10(5) uninfected peripheral blood mononuclear cells. Of the twelve participating laboratories, three reported correct results for the dilution series as well as for uninfected specimens. One or more classification errors were recorded for 12% of the samples for which the diagnosis was expected to be positive or negative. Samples containing 10 copies of the target template were correctly reported by eleven of the twelve participants. The average sensitivity was 97%. The results of the study revealed no significant differences between the Amplicor kit and in-house procedures. Most of the classification errors occurred in specimens from HIV-negative samples. Out of 36 negative samples, 5 were reported false positive, showing that contamination remains a problem for some laboratories, regardless of the PCR test performed. Careful laboratory techniques and internal as well as external quality control procedures will help avoiding carryover contamination.
DNA, Viral↗
Evolutionarily conserved positive and negative cis-acting elements control the blastoderm-specific expression of the Drosophila serendipity alpha cellularisation gene.
The serendipity alpha (sry alpha) cellularisation gene is only transcribed at the blastoderm stage, when this morphogenetic event takes place. We show that a 95 bp sry alpha upstream region is sufficient for blastoderm-specific expression of a lacZ reporter gene. This region encompasses four nucleotide motifs (I-IV, 5' to 3') conserved at similar relative positions in several Drosophila species. Removal of motif I leads to ectopic expression of lacZ in precursor cells of the PNS. Deletion of motif IV decreases the level of lacZ transcripts and modifies their banded pattern of accumulation late in cycle 14, whereas deletion of motifs II and III abolishes the sry alpha promoter activity. Motif III includes a consensus recognition site for b-HLH proteins. A point mutation in this E-box both severely reduces lacZ expression at blastoderm and prevents its ectopic expression in the PNS upon deleting motif I. These two effects depend upon da+ activity, suggesting that daughterless may positively control sry alpha transcription.
Animals↗
[Idiopathic CD4 lymphocytopenia with lethal Salmonella typhimurium sepsis].
In April 1991, a then 43-year-old woman fell ill with a systemic cryptococcal infection which responded well to antimycotic treatment. Four months later she was found to have a T-helper cell deficiency (48/microliters, 15%). Since August 1992 she noted an increased tendency towards infections and had recurrent fever bouts over 4 weeks, which led to her hospitalization in January 1993. Repeated HIV tests were negative. During the first 4 days she was free of infection, but developed a temperature of 39.1 degrees C on the fifth day, and within a few hours a fulminant septicaemia with cardiorespiratory failure developed ending fatally the same day. Blood cultures drawn during fever bouts grew Salmonella typhimurium by the time of her death. Lymphocyte differentiation revealed absolute and relative reduction in the number of CD4-lymphocytes (158/microliters; 18%), retrospectively providing the diagnosis of idiopathic CD4-lymphocytopenia.--In cases such as this there is the need of including opportunistic and masked disease entities in the differential diagnosis, even in the absence of HIV infections, and start early treatment.
Adult↗
AsnC, a multifunctional regulator of genes located around the replication origin of Escherichia coli, oriC.
The expression of the gidA gene which is located immediately counterclockwise of the replication origin of Escherichia coli, oriC, was found to be negatively regulated by the AsnC protein in an in vitro transcription-translation system. This effect is not due to simple repression of transcription originating at the gidA promoter, because the AsnC protein did not change the level of gidA promoter dependent transcription as analysed by promoter-galK fusions and by S1 mapping. From these data we conclude that the AsnC protein controls gidA gene expression at a post-transcriptional level. gidA is the third gene in the oriC region, besides asnA and asnC, whose expression is under AsnC control. However, the mechanisms involved are different: regulation of transcription in the case of asnA and asnC and post-transcriptional control of gidA. The gidA promoter was mapped by deletion analysis and by S1 mapping. We defined two regions that affect promoter activity negatively. Additional transcripts, regulated by AsnC, started more than 300 bp upstream of the gidA promoter and were found to enter the gidA region. These transcripts, originating either at the mioC and/or the ansC promoter traverse the replication origin.
Asparaginase↗
Transcription in the region of the replication origin, oriC, of Escherichia coli: termination of asnC transcripts.
Transcription from the asnC promoter was found to proceed through the replication origin, oriC, into the gidA gene of Escherichia coli. Between 10% and 20% of asnC transcripts reached oriC in vivo. Termination sites in the intergenic region between asnC and mioC and within mioC were determined in vivo and in vitro using S1 mapping. Only about 10% of the transcripts terminated at the asnC terminator in vivo. DnaA protein dependent termination was observed close to the binding site, dnaA box, for DnaA protein. In the in vitro replication system asnC transcripts did not reach oriC, suggesting that asnC transcripts are not involved in the initiation of replication, contrary to mioC transcripts. We suggest that oriC and mioC might have been transposed during evolution into an asnC regulation.
Bacterial Proteins↗
Transcripts within the replication origin, oriC, of Escherichia coli.
Transcription start and termination sites were mapped in the E. coli replication origin, oriC. Outward transcription from within oriC (promoters Pori-r and Pori-l) was found to start in vivo at position 178 for Pori-l and at positions 294 and 304 for Pori-r, respectively. These transcripts were terminated after 100-150 bases, at terminators designated Tori-l and Tori-r. Transcription from the 16 kd promoter, which lies clockwise adjacent to oriC and promotes transcription toward oriC, started at position 757 and gave transcripts with 3' ends at several positions within and to the left of the minimal replication origin. However, the majority of transcripts traversed the whole oriC region, and were not terminated within the DNA segment tested. Transcription of the chromosomal 16 kd gene was negatively regulated by DnaA protein and positively affected by dam methylation. The possible function of these transcripts is discussed.
DNA Replication↗
Reduced transcription of the rnh gene in Escherichia coli mutants expressing the SOS regulon constitutively.
We have analysed the transcription levels for the convergently overlapping Escherichia coli genes for the DNA polymerase III proofreading function (dnaQ) and ribonuclease H (rnh). The two tandem dnaQ promoters are about three times more active than the single rnh promoter as shown by analysing the level of in vivo transcription using dnaQ-galK and rnh-galK fusions. In E. coli mutants constitutively expressing the pleiotropic SOS response, which includes activities that enhance DNA repair, recombination and mutagenesis, a strong reduction in rnh transcription was observed. The lexA51 recA441 double mutant which fully expresses the SOS response shows the strongest reduction in rnh transcription and the highest increase in dnaQ transcription. Nuclease S1 mapping supported the finding that a constitutive expression of SOS function leads to a strong reduction in rnh transcription.
DNA Polymerase III↗
Regulation of transcription of the chromosomal dnaA gene of Escherichia coli.
By comparative S1 analysis we investigated the in vivo regulation of transcription of the chromosomal dnaA gene coding for a protein essential for the initiation of replication at the chromosomal origin. Inactivation of the protein in dnaA mutants results in derepression, whereas excess DnaA protein (presence of a DnaA overproducing plasmid) leads to repression of dnaA transcription. Both dnaA promoters are subject to autoregulation allowing modulation of transcriptional efficiency by at least 20-fold. Increasing the number of oriC sequences (number of DnaA binding sites) in the cell by introducing oriC plasmids leads to a derepression of transcription. Autoregulation and binding to oriC suggest that the DnaA protein exerts a major role in the regulation of the frequency of initiation at oriC. The efficiency of transcription of the dnaA2 promoter is reduced in the absence of dam methylation, which is involved in the regulation of oriC replication.
Bacterial Proteins↗
dnaA protein-regulated transcription: effects on the in vitro replication of Escherichia coli minichromosomes.
Both initiation of replication and initiation of transcription are influenced by dnaA protein, when minichromosomes are assayed in vitro for dnaA protein complementation. This dnaA protein effect is seen only if minichromosomes are used containing the 16-kd promoter, from which transcription is directed into the minimal origin. Determination of the 16-kd promoter activity both in vivo and in vitro showed that this strong promoter is specifically repressed by dnaA protein. The 16-kd promoter is thus an integral regulatory region of oriC.
Bacterial Proteins↗
The occurrence of two ribosomal ribonucleases depending on growth phase in yeast. Induction of ribonuclease in glucose-starved cells.
There are two latent ribonucleases associated with the 40 S subunits of yeast ribosomes which differ in their digestion products, pH optimum, molecular weight, and in their activity during growth phase. The 3'-nucleotide-producing enzyme is active only in the late logarithmic or stationary growth phase, whereas the ribonuclease which produces 5'-nucleotides is present at all growth phases. The enzymes were separated by affinity chromatography and were partially characterized. By changing growth conditions--i.e. decreasing and increasing the glucose concentration in the medium--the activity of the 3'-ribonuclease could be induced or reduced.
Cell Cycle↗
The induction of a ribosomal ribonuclease in Saccharomyces cerevisiae.
The ribosomal ribonuclease of yeast is induced after a withdrawal of glucose. Cycloheximide inhibits the synthesis of the enzyme under conditions of induction but antimycin shows no effect. Hence, the increase of the ribonuclease activity during growth of yeast is due to newly synthesized enzyme, and the induction does not depend on respiration.
Aerobiosis↗