Nucleotide sequence and expression of two beta-tubulin genes in Stylonychia lemnae.

The gene-sized macronuclear DNA of the hypotrichous ciliate Stylonychia lemnae contains one size class of DNA molecules of 1.85 kb (1 kb = 10(3) base-pairs) coding for beta-tubulin. These DNA molecules consist of two different beta-tubulin genes, beta 1 and beta 2, which are amplified to about 150,000 (beta 1) and 30,000 (beta 2) copies per macronucleus. Both genes were cloned and sequenced entirely. The coding sequences of the two molecules (1329 base-pairs including TGA) predict identical amino acid sequences for the proteins and show a nucleotide homology of 97.2%. The nucleotide as well as the encoded amino acid sequences are highly conserved, when compared to beta-tubulin genes from vertebrates. The ciliate-specific codon TAA specifying glutamine is present only in the beta 2-tubulin gene, whereas glutamine is encoded soley by CAA in the beta 1-tubulin gene. The 5' and 3'-non-coding regions of both beta-tubulin genes are similar in length, but differ extremely in nucleotide sequence. Both beta-tubulin genes are transcriptionally active in S. lemnae, although not all putative transcription-regulatory sequences known from higher eukaryotes can be detected within the non-coding regions. The two transcription products localized by S1-mapping experiments show a similar length of about 1.40 kb and transcription seems to be regulated differently for beta 1 and beta 2.

Transfection of the hypotrichous ciliate Stylonychia lemnae with linear DNA vectors.

We present a new protocol for the transfection of Stylonychia lemnae with linear DNA vectors containing the neomycin-resistance gene from Escherichia coli transposon Tn5. The taking up of heterologous DNA is achieved by damaging the cells' protein coat with urea prior to transfection according to the calcium phosphate co-precipitation procedure. After transfection, transformed cells can be enriched by selection with the antibiotic drug G418. Hybridization experiments show that macronuclear DNA of these G418-selected cells contains molecules homologous to the transfected vector DNA, which are altered by some recombination process. Transfected cells, which have grown for more than 100 cell cycles in antibiotic-free medium, still contain vector-homologous DNA, but recombination continued during this time. We are able to transfect Stylonychia early after conjugation, a process which is followed by complex genome rearrangements and amplification. In these experiments we observed considerable amplification of vector-homologous DNA molecules as compared to transfected vegetative cells. Again these molecules are altered by recombination in respect to the original vector DNA. As soon as suitable vectors are available, the transfection protocol presented here can be a basic tool for the study of DNA replication, transcription and macronuclear development in Stylonychia.

A comparative study of RNAs from four ciliate groups and a mollusc and data on their poly(A)(+) RNA in vitro translation.

RNAs from the ciliates Euplotes octocarinatus, Paramecium tetraurelia, Stylonychia lemnae, Tetrahymena thermophila and the snail Lymnaea stagnalis were isolated and compared for structural differences. The organisms were found to differ considerably in the size and structure of their rRNAs, the extent of polyadenylation and the length of the respective poly A tails of their mRNAs. The mRNAs of Euplotes, Paramecium, Stylonychia and Lymnaea were also compared for in vitro translatability in the rabbit reticulocyte lysate and the wheat germ translation systems. In accordance with earlier observations for Paramecium [15] and the finding that Paramecium, Stylonychia, Oxytricha and Tetrahymena deviate from the universal genetic code by using the stop codons UAA and UAG to encode glutamine [4, 8,11,12,21] it was found that Paramecium and Stylonychia poly(A)(+) RNAs were poor templates for in vitro translation. Unexpectedly, however, poly(A)(+) RNA of the ciliate Euplotes was readily translated in vitro and yielded a great number of polypeptides with different molecular weights comparable to the data obtained with poly(A)(+) RNA of Lymnaea.

Regulatory structures of gene expression, DNA-replication and DNA-rearrangement in macronuclear genes of Stylonychia lemnae, a hypotrichous ciliate.

Seven transcriptionally active macronuclear DNA molecules of Stylonychia lemnae have been analyzed for potential regulatory sequences of gene expression, DNA replication and DNA rearrangement. Transcription initiation is mediated by a canonical TATA-box or by TATA-box-like sequences, and transcription efficiency may be regulated by gene-specific downstream promoter elements (DPE). Putative signals for RNA 3'-formation are represented by noncon-served palindromes located upstream of the mature RNA 3'-termini. Sequences indicating polyadenylation of the mRNA molecules are not detectable. A single palindrome of A- and T-residues is present within the 80 5'-terminal bases of each macronuclear DNA molecule and very likely functions as the replication origin. The 7 DNA molecules consist of several nonconserved inverted and direct repeats (IR, DR) suggested to play a role in DNA rearrangement during macronuclear development.

Nucleotide sequence of a macronuclear DNA molecule coding for alpha-tubulin from the ciliate Stylonychia lemnae. Special codon usage: TAA is not a translation termination codon.

The gene-sized macronuclear DNA of the hypotrichous ciliate Stylonychia lemnae contains two size classes of DNA molecules (1.85 and 1.73 kbp) coding for alpha-tubulin. Each macronucleus contains about 55000 copies of the 1.85 kbp molecules and about 17000 copies of the 1.73 kbp DNA molecules. Five macronuclear molecules of these sequences were cloned and sequenced, one, from the 1.85 kbp size class in its entirety. The 5 sequences fell into two classes suggesting that Stylonychia lemnae contains at least two different alpha-tubulin genes. All 5 clones show the codon TAA in the same nucleotide positions of the coding region. In this position the TAA codon cannot function as a translational stop codon and we suggest that this codon codes for the amino acid glutamine. The nucleotide sequence of the coding region as well as the encoded amino acid sequence is highly conserved compared to alpha-tubulin genes from vertebrates. The noncoding regions show several putative transcription-regulatory sequences as well as sequences presumably functioning as replication origins.

Both alpha-tubulin genes are transcriptionally active in Stylonychia lemnae.

Macronuclear DNA of the hypotrichous ciliate Stylonychia lemnae contains two size-classes of molecules coding for alpha-tubulin. Analysis of their coding regions demonstrates that these two size-classes represent two different functional alpha-tubulin genes, alpha 1 and alpha 2; a comparison of these regions shows a 97% homology in their nucleotide sequence and 98.5% in their amino acid sequence. S1-mapping experiments indicate that both alpha 1- and alpha 2-tubulin genes are transcribed. The 5' and 3' noncoding regions flanking the alpha 1- and alpha 2-tubulin genes differ in both length and nucleotide sequence within one strain. When different strains are compared, however, identical non-coding regions and slightly varying coding regions can be found in DNA molecules containing the alpha 1-tubulin genes.

[Laboratory diagnosis of parvovirus B 19 infection: comparison of ten commercial IgG- and IgM-antibody tests]. / Labordiagnostik von Parvovirus-B19-Infektionen: Vergleich von zehn kommerziellen IgG- und IgM-Antikörpertests.

Seven ELISAs, two westernblots and one immunofluorescence assay for serological detection of parvovirus-B19-infection were compared with regard to their sensitivity and specificity. All ten assays are commercially available and use recombinant proteins (VP1, VP2) from bacterial and eukaryotic expression systems and synthetic viral peptides as antigens. The ELISA assays comprise indirect ELISA and mu-capture systems. All assays were tested for their sensitivity with follow-up sera from 21 patients with acute parvovirus B19 infections. The specificity was analysed by 194 sera from pregnant women and from patients with acute other virus infections or rheumatoid factors, but without acute parvovirus infection. Only three ELISAs, both westernblots and the immunofluorescence assay can be recommended for the IgG-measuring (prevalence). For the detection of an acute parvovirus infection, especially during pregnancy, two ELISAs show good IgG- and IgM-results, one ELISA is less recommendable and all other assays show only a poor IgM-specificity.

Oxygen dependence of nuclear DNA replication in Ehrlich ascites cells.

Oxygen was excluded from cultured Ehrlich ascites cells for 5-7 h and then readmitted. During the anaerobic period and for about 1 h following reoxygenation the DNA synthesis of the cells was studied by determining the DNA synthesis rate from [3H] thymidine incorporation data, by evaluation of the thymidine (pulse labelling) index, by DNA fibre autoradiography, and by alkaline sucrose gradients in order to follow the maturation of the daughter chains. The DNA synthesis rate was found to decay to a few percent of the initial value within 5-7 h after deoxygenation. Immediately after reoxygenation it increased to exceed the control level within 0.5-1 h. The only partial process of the genome replication definitely responding to deoxygenation/reoxygenation was the initiation of new replicon units, while progress of the replication forks and maturation of the new daughter chains were not significantly affected. The coordination of replicon initiation within groups or clusters was maintained throughout. The interruption of replication at the level of initiation of clusters upon deoxygenation was interpreted as a regulatory response of the cells to ensure basic viability under unfavourable conditions.

Prevalence of antibodies to human herpesvirus 6 in different age groups, in children with exanthema subitum, other acute exanthematous childhood diseases, Kawasaki syndrome, and acute infections with other herpesviruses and HIV.

We determined IgG antibodies against Human Herpesvirus-6 (strain Uganda 1102, M. D. Griffin, London) in the indirect immunofluorescence test in sera from 1105 persons of various age groups. Of these sera 570 were retested using HHV-6 strain St. W. (Prof. Schneweis, Bonn). We could confirm that maternal antibodies decrease between birth and six months of age and the seropositive rate rises rapidly between seven months and five years of age up to 79.5%. Between six and ten years and up to 40 years, the antibody-positive rate lies around 81.3% and 66%, respectively. To confirm the causal nature of human herpes virus type 6 (HHV-6) for exanthema subitum we could demonstrate eight seroconversions testing sera from 14 patients with roseola infantum. In addition, the virus was isolated from peripheral blood lymphocytes of children during the acute fever phase in four cases in tissue culture and in six cases the virus was detected by positive hybridization. In single and some paired sera from patients with acute exanthematous diseases, rubella (n = 28), parvovirus B 19 (n = 24), measles (n = 17), mumps (n = 27), adenovirus (n = 27) and parinfluenza virus type 3 (n = 28) and in sera from patients with Kawasaki syndrome (n = 20), acute varicella-zoster- (n = 27), acute herpes simplex- (n = 18) and HIV-1 infection (n = 50), we found no HHV-6 IgM antibodies and no HHV-6 IgG antibody rises. We could only demonstrate an HHV-6 seropositive rate according to our age-prevalence study.(ABSTRACT TRUNCATED AT 250 WORDS)

Virus detection in the fetal tissue of a premature delivery with a congenital varicella syndrome. A case report.

Cases of congenital varicella syndrome have been published, to date, a single case reports. Isolation attempts of Varicella-Zoster virus from fetal tissues have, thus far, been unsuccessful. This is a first report of detection of Varicella-Zoster virus in fetal tissue by means of DNA hybridization technique in a typical case of congenital varicella syndrome in a premature delivery of the 27th gestational week. The case is documented anamnestically, sonographically, pathologically and virologically. In women with primary varicella infection during pregnancy good sonographical controls are recommended. In cases with sonographically characteristical signs prenatal diagnosis with puncture of the umbilical vein cord and placentocentesis may be considered. The varicella DNA detection should be supplemented, however, by the polymerase chain reaction.

Identification of an amplification promoting DNA sequence from the hypotrichous ciliate Stylonychia lemnae.

The macronucleus of the hypotrichous ciliate Stylonychia lemnae contains a 1218 bp long DNA molecule which becomes highly amplified during vegetative growth due to a continuous overreplication over a long time range. The region which is located upstream the open reading frame of the overamplified 1.2kbp Stylonychia DNA molecule enabled plasmids containing an inefficiently transcribed thymidine kinase gene to persist and amplify upon transfection into mouse L fibroblasts under selective conditions. This region contains long AT-rich stretches. The AT-rich sequences interact with a previously characterized HMG-I like protein from mouse Ehrlich ascites tumour cells. A binding activity for AT-rich stretches could also be identified in macronuclear extracts from Stylonychia lemnae. We suggest a common mechanism for overamplification in Stylonychia macronuclei during vegetative growth and amplification of plasmid DNA in heterologous mouse cells under the influence of a common element.