Ewald Beck

Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5

The nucleotide sequence of 1200 bp from the unique region of transposon Tn5 containing the neomycin phosphotransferase gene (neo) was determined, and the location of the neo gene was identified by deletion mutants in a translational reading frame of 792 bp. The derived gene product, an aminoglycoside 3-phosphotransferase (APH) II, consists of 264 amino acid residues and has a calculated Mr of 29053. Its amino acid sequence shows sequence homologies to the APH type I enzyme coded for by transposon Tn903 (Oka et al., 1981).

Primary structures of Sm31/32 diagnostic proteins of Schistosoma mansoni and their identification as proteases

We have constructed cDNA clones containing the complete nucleotide sequences coding for two highly antigenic Schistosoma mansoni adult worm proteins, Sm31 and Sm32. The predicted amino acid sequence of Sm31 shows significant homology to mouse, rat and human cathepsin B. The nucleotide sequence of Sm32 is identical to that reported by others for S. mansoni haemoglobinase. The different nucleotide sequences demonstrate the existence of two different proteolytic enzymes, both of which are synthesised in the form of precursor molecules. Structural homology of the schistosome cathepsin B to the mammalian ones indicates that the mature protein is processed from a propeptide. The calculated molecular weight of haemoglobinase of 47,000 suggests that post-translational processing is also involved in generating an active protease.

Nucleotide sequence and genome organisation of filamentous bacteriophages f1 and fd

Abstract The DNA sequence of the filamentous phage f1, consisting of 6407 nucleotides, has been determined. When compared with the DNA sequence of the related filamentous phage fd (Beck et al., 1978), the f1 sequence is one nucleotide shorter and differs in 180 positions from the fd DNA. Only ten of these base exchanges cause amino acid exchanges in the known gene products. Most of the exchanges in f1 are the same as in M13 (Van Wezenbeek et al., 1980), showing a near identity of these two phage (there are only 59 nucleotide differences). Regulatory units for replication, transcription, and translation are in their essential parts identical in all three phage.

Fixation of mutations in the viral genome during an outbreak of foot-and-mouth disease: heterogeneity and rate variations

Rates of fixation of mutations during the evolution of the foot-and-mouth disease virus (FMDV) C1 in nature have been estimated by hybridization of viral RNA to cloned cDNAs representing defined FMDV genome segments, and comparison of the selected RNAs by T1 RNase oligonucleotide fingerprinting. Values ranged from less than 0.04 X 10(-2) to 4.5 X 10(-2) substitutions per nucleotide per year (s/nt/yr), depending on the time period and the genomic segment considered. Rates for viral structural protein genes were up to sixfold higher than for nonstructural protein genes. Values in excess of 10(-2) s/nt/yr have been measured for the RNA region that encodes VP1-VP3. The nucleotide sequences of the major immunogenic region of capsid protein VP1 have been determined for six new FMDV C1 isolates, and they are compared with the two previously known sequences of FMDV C1 (C-S8 and C1-O). Both oligonucleotide fingerprinting of selected RNA fragments and direct nucleotide sequencing demonstrate that genetic heterogeneity exists among three viruses isolated on the same day, introducing a significant indetermination in the evaluation of fixation rates of mutations. During the FMDV C1 outbreak, amino acid substitutions did occur that are known to affect the immunological properties of the virus. The proportion of mutations between two viral RNAs does not increase significantly with the time elapsed between the two isolations, suggesting a cocirculation of multiple, related, nonidentical FMDVs (evolving quasispecies) as the mode of evolution of this agent.

Cloning of diagnostic 31/32 kilodalton antigens of Schistosoma mansoni

A cDNA library derived from messenger RNA of adult Schistosoma mansoni was constructed in lambda gt11 and schistosome antigens were expressed as fusions with the amino terminus of the beta-galactosidase of Escherichia coli. Using mouse and human infection sera, recombinant clones specific for a 31/32 kDa doublet having a potential in the immunodiagnosis of schistosomiasis were selected. The specificity of the clones was verified by their reactivity with monoclonal antibodies. The identity of the cloned epitopes with those of the native proteins was confirmed by Western blot analysis of total schistosome proteins, using both antibodies affinity purified from the fusion proteins and antisera raised in rabbits against the fusions. The reactivity of the cloned antigens with human infection sera suggests their usefulness for the immunodiagnosis of human schistosomiasis.

Cloned structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12

SummarypTU 100 is a hybrid plasmid constructed by cloning a 7.5 Kb EcoRI fragment (carrying the wildtype ompA gene) onto pSC 101 (Henning et al., 1979). This plasmid confers sensitivity to phages Tull* and K3h1 when present in an ompA host strain, due to the expression of the phage receptor protein II* from the plasmid ompA+ gene. Plasmid mutants have been isolated that have become resistant to one or both of these phages. Restriction endonuclease analysis and DNA-sequencing studies in these plasmids demonstrate that a BamHI site and two PvuII sites are located within the ompA gene. BamHI cuts the gene at a site corresponding to residue 227 within a total of 325 amino acid residues.Neither the wildtype ompA gene nor the BamHI fragment encoding the NH2-terminal part of the protein (residues 1–227) could be transferred to a high copy number plasmid, presumably due to lethal overproduction of the protein or its NH2-terminal fragment. However, the NH2-terminal fragment derived from one of the ompA mutants of pTU100 could be transferred to the high copy number plasmid pBR322, and was expressed in the presence of the amber suppressors supD or supF. Under these conditions two new envelope proteins with apparent molecular weights of 30,000 and 24,000 were synthesized, and the cells became sensitive to phage TuII*, indicating the presence of phage receptor activity in the outer membrane. The major, 24,000 dalton protein has the molecular weight expected of a protein comprising residues 1–227 of protein II*. DNA-sequencing studies demonstrated that no termination codons are present in the DNA region immediately downstream from the BamHI site at residue 227 in this hybrid plasmid, and it is therefore likely that the 24,000-dalton protein arises from the posttranslational proteolytic cleavage of a larger polypeptide. The 30,000-dalton protein is a likely candidate for such a larger polypeptide. These results also demonstrate that the 98 CO2H-terminal residues of wildtype protein II* (resisdues 228–325) are not required either for the activity of the protein as a phage receptor or for its incorporation into the outer membrane.

Specificity of σ-dependent binding of RNA polymerase to DNA

SummaryAlthough a large number of E. coli RNA polymerase molecules can bind to phage T3 DNA, not more than three remain bound per DNA template after addition of poly inosinic acid (poly I) which has a high affinity for the enzyme. These stable complexes are able to initiate RNA chains without lag as the enzyme is resistant against rifampicin if substrate is added simultaneously with the drug. Poly I resistant complexes decay very rapidly in the cold (Fig. 2) and are not formed in the absence of the polymerase σ factor (Table 2). The data provide additional support for the idea that the σ factor effects the binding of the enzymes to specific sites on the DNA template.

The nucleotide sequence of a DNA fragment from the replication origin of the antibiotic resistance factor R1drd19

SummaryThe recombinant plasmid pRK101 contains a DNA fragment which carries the complete replication origin of the antibiotic resistance factor R1drd-19 inserted into the vector plasmid pBR322. In a spontaneously arising mutant of this plasmid (pRK 103) a deletion of about 215 base pairs (bp) has been detected by heteroduplex analysis and mapping with restriction endonucleases. Essential parts of the replication origin must be located in the deleted sequence. The deletion mutant pRK103, in contrast to its parent plasmid pRK101 is not replicated under the control of the R1 replicon, even when the R1 factor or copy mutants of it are present within the same cell. These latter plasmids can complement a plasmid-specific protein not coded by pRK101 but essential for R1-directed replication. The nucleotide sequence of a 252 bp HpaII fragment covering about 170–200 bp of the deletion was determined. This piece of DNA is rich in G and C and contains a series of small palindromes, symmetrically arranged repeated sequences and short selfcomplementary structures which may be of significance for the initiation of the DNA replication. The possibility that the sequenced DNA fragment comprises a major part of the replication origin of R1drd-19 is discussed.

Sequence and Regulatory Signals of the Filamentous Phage Genome

The genome of the male-specific filamentous coliphages fd, f1, and M13 is a circular single-stranded DNA of approximately 6400 bases, which codes for at least eight genes (Marvin and Hohn 1969; Ray 1977). Upon infecting a cell, a double-stranded replicative-form (RF) DNA is formed, and this RF DNA then serves as a template for mRNA synthesis as well as for progeny phage DNA production. Both reactions are catalyzed and controlled largely by host proteins. Therefore, the structure and function of the filamentous phage DNA have been studied extensively as a model system for elucidating the molecular mechanisms involved in DNA replication and gene expression. The first nucleotide sequences of filamentous phage DNA to be determined were the pyrimidine tracts obtained by the Burton and Peterson depurination procedure; tracts of up to 20 bases in length were sequenced (Petersen and Reeves 1969; Ling 1972). Oligonucleotide-primed DNA synthesis was used by Oertel and Schaller (1972) to determine the order of pyrimidine tracts in a pyrimidine-rich segment of fd DNA (positions 5539–5630, Fig. 1) and, in combination with ribosubstitution, by Sanger et al. (1973 Sanger et al. (1974) to deduce the sequence of 85 nucleotides in f1 DNA (positions 6303–6387, Fig. 1). Three ribosome-binding sites on DNA-directed mRNA were sequenced by Pieczenik et al. (1974). Ribosomes bind specifically to the start point of translation on mRNA, and the bound region can be isolated and sequenced after RNase treatment. Similar protection experiments with RNA polymerase allowed the isolation and sequencing of an RNA-polymerase-binding site from...

Expression of diagnostic 3132 kilodalton proteins of Schistosoma mansoni as fusions with bacteriophage MS2 polymerase

An expression plasmid pEx34b was used to synthesise parts of the 31/32 kDa Schistosoma mansoni antigens as fusions with the amino terminus of the phage MS2 polymerase. Purified MS2-schistosome fusion proteins reacted specifically in an enzyme-linked immunosorbent assay with sera from S. mansoni-infected patients. The observation that the majority of human sera tested recognised schistosome-specific epitopes, but not the MS2 polymerase fragment, suggests that the fusion proteins are useful for the immunodiagnosis of schistosomiasis and might be incorporated in a serological test system based on recombinant antigens.