Wolfgang Rüger

T4 early promoter strength probed in vivo with unribosylated and ADP-ribosylated Escherichia coli RNA polymerase: a mutation analysis.

The consensus sequence of T4 early promoters differs in length, sequence and degree of conservation from that of Escherichia coli sigma(70) promoters. The enzyme interacting with these promoters, and transcribing the T4 genome, is native host RNA polymerase, which is increasingly modified by the phage-encoded ADP-ribosyltransferase, Alt. T4 early transcription is a very active process, possibly out-competing host transcription. The much stronger T4 promoters enhance viral transcription by a factor of at least two and the Alt-catalysed ADP-ribosylation of the host enzyme triggers an additional enhancement, again by a factor of about two. To address the question of which promoter elements contribute to the increasing transcriptional activity directed towards phage genes, the very strong E. coli promoter, Ptac, was sequentially mutated towards the sequence of the T4 early promoter consensus. Second, mutations were introduced into the highly conserved regions of the T4 early promoter, P8.1. The co-occurrence of the promoter-encoding plasmid pKWIII and vector pTKRI, which expresses Alt in E. coli, constitutes a test system that allows comparison of the transcriptional activities of phage and bacterial promoters, in the presence of native, or alternatively ADP-ribosylated RNA polymerase. Results reveal that T4 early promoters exhibit a bipartite structure, capable of strong interaction with both types of RNA polymerase. The -10, -16, -42 and -52 regions are important for transcript initiation with the native polymerase. To facilitate acceleration of transcription, the ADP-ribosylated enzyme requires not only the integrity of the -10, -16 and -35 regions, but also that of position -33, and even more importantly, maintenance of the upstream promoter element at position -42. The latter positions introduced into the E. coli Ptac promoter render this mutant promoter responsive to Alt-ADP-ribosylated RNA polymerase, like T4 early promoters.

Alternative splicing in PAX2 generates a new reading frame and an extended conserved coding region at the carboxy terminus

Abstract PAX2 is a member of the PAX multigene family encoding transcription factors active in specific tissues during embryogenesis. Several PAX/Pax genes (PAX and Pax describe homologous genes in human and mice, respectively) have been shown to possess critical morphogenetic functions as identified by the analysis of mice targeted for Pax genes and the phenotype of patients heterozygous for PAX mutations. Mutations in PAX2 have been shown to be implicated in independent cases of renal-coloboma syndrome. Here, we report the characterisation of a new PAX2 isoform, viz. PAX2d, which arises because of the use of an alternative acceptor splice site within exon 12 of the PAX2 gene; this leads to a shift in the reading frame. A conserved coding region extended over the regular stop codon may emphasize the biological significance of this isoform.

ModA and ModB, two ADP-ribosyltransferases encoded by bacteriophage T4: catalytic properties and mutation analysis.

Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB. These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins. In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes. Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes. In vitro transcription assays performed with Alt- and ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced. The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control. In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.

Characterization of cDNA clones encoding a major microneme antigen of Sarcocystis muris (Apicomplexa) cyst merozoites

Two monoclonal antibodies directed against a microneme antigen of Sarcocystis muris cyst merozoites (16/17 kDa band doublet) were used to isolate cDNA clones from a lambda ZAP expression library. Restriction analysis revealed that the inserts were highly similar, with sizes ranging between 1.8 and 2.3 kb. In addition, a full-length cDNA insert of 2.6 kb was obtained by hybridization screening. On Northern blots, a single mRNA species of 2.7 kb was detected by a cDNA-derived probe. Southern blot hybridization suggests that the gene is present as a single copy. The nucleotide sequence of the full-length clone contains a single reading frame with a coding capacity of 26.5 kDa. The hypothetical polypeptide consists of a putative N-terminal signal peptide followed by a hydrophilic domain of unknown function, and the mature protein sequence. After purifying the 16/17 kDa antigen from cyst merozoites, a partial N-terminal amino acid sequence was obtained. Thus, the identity of the cDNA sequence was confirmed. The deduced sequence of the mature protein is predominantly hydrophilic and rich in cysteine (8.7%). Database searching suggested weak homologies of the hypothetical polypeptide to plasma kallikrein, tenascin and blood coagulation factors.

Chemical and Enzymatic Synthesis of DNA Fragments Containing 5-(β-D-Glucopyranosyloxymethyl)-2′-deoxycytidine − a Modified Nucleoside in T4 Phage DNA

DNA fragments 23 and 25, containing 5-hydroxymethyl-2′-deoxycytidine (5-HMdC) and 5-(β-D-glucopyranosyloxymethyl)-2′-deoxycytidine (5-GlcMdC) units, respectively, were assembled by means of a solid support-assisted synthesis using phosphoramidite building blocks 4 and 5, respectively. In addition, 20-mer 23 was converted into fragment 26 by a glycosylation with uridine diphosphoglucose (UDPG), catalyzed by T4 β-glucosyltransferase (BGT).

A physical map of bacteriophage T4 including the positions of strong promoters and terminators recognized in vitro

SummaryWe present a linearized physical map of the genome of bacteriophage T4. This map contains the cleavage sites for restriction enzymes SmaI, KpnI, SalI, BglII, XhoI, XbaI, ClaI, HaeII, EcoRI, and EcoRV. It also contains about 200 TaqI sites. The promoter sites recognized in vitro and a number of rho independent terminators have also been mapped.

Transcription of RNA polymerase binding sites isolated from T4 phage DNA.

Abstract RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, DNA-dependent, EC 2.7.7.6) was bound to T4 DNA. After extensive digestion with deoxyribonuclease, DNA regions protected by the polymerase against the nucleolytic action were isolated. The template activity of these DNA fragments in the RNA polymerase reaction was studied by varying the salt concentrations and by the addition of deoxyribonuclease, ribonuclease, rifampicin and streptolydigin 1. 1. These DNA fragments serve as templates for RNA transcription. The reaction is not markedly influenced by the KCl concentration. Once the polymerase is bound to the isolated DNA fragments further addition of deoxyribonuclease does not reduce the incorporation of ribonucleotides. Rifampicin, streptolydigin or the lack of one of the four ribotriphosphates stops the reaction. 2. 2. The transcription product is composed of AMP, CMP, GMP and UMP. From hybridization experiments the chain length of the synthesized RNA was estimated to be 15–20 ribonucleotides. The transcription product hybridizes specifically with T4 DNA.

Deoxyuridylate-hydroxymethylase of bacteriophage SPO1

Phage SPO1 of Bacillus subtilis carries hydroxymethyl-deoxyuridylate in place of thymidylate in its DNA. The enzyme, responsible for the conversion of dUMP to HmdUMP, is a dUMP hydroxymethylase, encoded by the SPO1 gene 29. Here we describe the cloning and sequencing of the gene and the overexpression of the gene product. DNA hybridization using the DNA of bacteriophage T4 dCMP-hydroxymethylase gene as a probe, allowed us to identify and map g29 on a 3.9-kb restriction fragment, EcoRI*11. We determined the nucleotide sequence. One of the open reading frames detected, coding for a putative 44.6-kDa protein, showed significant amino acid homologies with all known thymidylate synthases. Gp29 was overexpressed in the pT7 system. Extracts prepared from induced cells show hydroxymethylase activity in a tritium release assay.

Cloning and expression in Escherichia coli of cDNAs encoding a 31-kilodalton surface antigen of Sarcocystis muris

Using polyadenylated RNA isolated from Sarcocystis muris cyst merozoites, we have constructed a cDNA library in the expression vector lambda ZAP. Immunoscreening with monoclonal and polyclonal antibodies directed against a 31-kDa surface antigen of S. muris [1] yielded a number of clones with insert sizes ranging between 1.1 kb and 1.3 kb. An additional clone with an insert length of 1.55 kb was isolated by screening with a labeled DNA probe derived from one of the cDNA clones. The cDNA sequence was found to contain an open reading frame specifying a polypeptide of 280 amino acids with a predicted size of 29.7 kDa. The deduced amino acid sequence is rich in serine and threonine (22%) and harbors a hypothetical N-terminal signal peptide sequence as well as a C-terminal glycosyl phosphatidylinositol anchor attachment site. The predicted amino acid sequence has been confirmed by peptide sequencing and an analysis of the overall amino acid composition of the 31-kDa protein. A recombinant protein was obtained which was recognized by the polyclonal antibodies directed against the 31-kDa antigen. Antiserum raised against the purified fusion protein specifically reacted with a 31-kDa protein from S. muris cystozoites. Southern blot analysis indicated that the corresponding gene exists as a single copy within the S. muris genome.

Physical mapping of the restriction fragments obtained from bacteriophage T4 dC-DNA with the restriction endonucleases SmaI, KpnI and BglII.

SummaryThe cytosine-containing DNA of a mutant of bacteriophage T4 was digested with restriction endonucleases SmaI, KpnI and BglII producing 5, 7 and 13 fragments respectively. Complete physical maps of the T4 genome were constructed with the enzymes SmaI and KpnI and an almost complete map with the enzyme BglII.