Walter Sauerbier

Structure and transcription of the gene for translation elongation factor 1 subunit alpha of zebrafish (Danio rerio)

The zebrafish gene for translation elongation factor 1 alpha (EF1 alpha) was isolated from a phage Lambda genomic library and sequence and structure determined. One gene copy of EF1 alpha per haploid set of chromosomes was found and no processed pseudogenes. A highly active promoter region was localized to a 277 bp PstI/PvuII fragment beginning 240 bp upstream from the tsp, but no transcription enhancing, or silencing activity was observed within 1 kbp upstream, or downstream from the promoter. Expression of EF1 alpha appears to be developmentally regulated.

Genomic studies of the Lucké tumor herpesvirus (RaHV-1)

Abstract Ranid herpesvirus 1 (RaHV-1) is the etiological agent of the Lucké renal adenocarcinoma of the North American leopard frog Rana pipiens. Construction of cosmid libraries containing RaHV-1 DNA inserts allowed the derivation of a BamHI map for the viral genome. Summation of fragment sizes indicates that the genome is 217u2009kbp in size, a value in accordance with the most recent published estimate (220u2009kbp) obtained by field-inversion gel electrophoresis. The DNA sequence of the 39,757-bp insert in 1 cosmid (cos54) was determined and was predicted to contain 21 complete and 3 partial genes. In all, 12 genes have distant counterparts in a fish herpesvirus (ictalurid herpesvirus 1) and are present in 2 blocks, 1 of which is relatively inverted. This indicates that RaHV-1 belongs to the fish virus lineage of the herpesvirus family rather than to the lineage populated by mammalian and avian viruses. The remainding 12 genes in cos54 lack counterparts in any other herpesvirus. One of these encodes a putative DNA (cytosine-5) methyltransferase. This raises the possibility that biological processes induced in the host by RaHV-1 might involve methylation of cellular DNA by the viral enzyme.

Flanking AT-rich sequences may lower the activation energy of cruciform extrusion in supercoiled DNA

In the absence of flanking AT-rich segments, cruciform transition energies of DNA palindromic sequences of random base composition are high and mainly dependent upon the base-stacking and -pairing parameters of the palindromic segment. When AT-rich sequences adjoin palindromes, the transition energy of cruciform extrusion is significantly lowered. An inverse relationship exists between the length of the AT-rich stretch and the cruciform transition energy. Long stretches lower the transition energies more than short stretches. At physiological salt and temperature conditions, equilibrium between cruciform extrusion and absorption for the inverted repeat sequences IRS-B and IRS-C of pBR322 derived plasmids is reached in less than five minutes.

Sequence Instability in the Long Terminal Repeats of Avian Spleen Necrosis Virus and Reticuloendotheliosis Virus

SummarySequence divergence between the 3′ long terminal repeats (LTR) of avian reticuloendotheliosis virus (REV), deletion variant proviral clone 2-20-4, and spleen necrosis virus (SNV)—proviral clones 14-44, 60, and 70—was found to involve two classes of base substitutions: low-frequency interspersed and high-frequency clustered substitutions. Clones 2-20-4 and 14-44 have diverged 4.4% owing to low-frequency substitutions. In contrast, two high-frequency substitution segments have diverged by 30% and 29%, respectively. Clustered substitutions appear to be located either within or next to tandem repeats, suggesting their introduction concomitant with sequence deletions and duplications commonly associated with such repeats. A new 19-bp tandem repeat is found in clone 2-20-4. Its sequence could have evolved from the 26-bp repeats found in the SNV clones.

Interactions between structure transitions in a torsionally constrained DNA

We used S1 nuclease cleavage in conjunction with gel electrophoresis to evaluate torsion-induced cruciform extrusion at two inverted repeat sequences, IRS-B and IRS-C of plasmid pUC12. These structure transitions affect each other through competition for the available torsional free energy according to their relative energies of activation and the magnitude of DNA duplex unwinding associated with each transition. They can be modulated by the level of DNA negative torsion. Interplays between transition sequences occur over long distances and are independent of relative orientation of transition sites. DNA binding factors that enhance or repress structural transitions of specific sequences may, thus, regulate the structural and functional properties of torsionally coupled, distal sequences.

pUC12-Wl: a plastnid vector for the study of cloned DNA conformation

pUC12-W1 is a new cloning vector for the study of torsion-induced structural transitions of insert DNA. It was derived from pUC12 by deleting three A + T-rich sequences which can undergo structural transitions when torsionally stressed. Transitions at these sites have low energy of activation and undefined structures. They complicate studies on transitions of DNA inserts by diverting torsional force and causing the vector to be undefined in helical and energetic terms. The new vector pUC12-W1, from which these segments have been deleted, will facilitate studies of torsion-induced structural transitions of insert DNA.

Mapping structurally perturbed sites in DNA by replication arrest and run-off replication

We describe a technique for rapid fine mapping of sites of torsion-induced perturbations of DNA structure. The technique involves strand scission or chemical base modification at structurally perturbed sites, replication arrest in a double-strand DNA sequencing reaction, and size analysis of replication products by electrophoresis on sequencing gels. Besides being less complicated and faster than site identification by conventional end-labeling methods, the technique assures high sequence specificity through the use of oligomeric sequencing primers. This property should be useful for in vivo mapping of DNA structural perturbations with known sequence within complex genomes.

Pyrimidine dimer dependent cleavage of single-stranded DNA by T4 UV endonuclease

T4 UV endonuclease cleaves double- and single-stranded DNA with equal specificity for photo-pyrimidine dimers. Thus, the enzyme can be used for mapping and quantifying pyrimidine dimers in single-stranded DNA as well as in double-stranded DNA. Mapping of pyrimidine dimers shows that rates of UV-dimerization are not only affected by 5, 3 adjacent bases, but also by position within pyrimidine tracts. Di-pyrimidines at 3 ends of tracts are more photoreactive than those at 5 ends.