Robert M. Benbow

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found.

Molecular analysis of transgenic plants generated by microprojectile bombardment: effect of petunia transformation booster sequence.

Supercoiled plasmid expression vectors containing the petunia transformation booster sequence (TBS) were introduced by microprojectile bombardment into dicotyledenous (tobacco) and monocotyledonous (maize) cells. TBS effected a 7.8- to 16-fold increase in transformation frequencies in tobacco, and a 1.7- to 2.4-fold increase in maize. Although TBS contains a well-defined transcription enhancer element, no increases in plasmid gene expression were observed. TBS did not alter integration patterns in transformants, and did not affect segregation of linkage in R1 progeny. Computer analyses of the TBS sequence revealed numerous modular elements previously shown to be associated with putative chromosomal replication origin regions in eukaryotes, including DNA unwinding elements, scaffold-associated regions and pyrimidine tracts.

High-resolution chromatography of nucleic acids on the gen-pak fax column

High-performance liquid chromatography (HPLC) on a Gen-Pak FAX column has been used to separate and purify microgram amounts of single- and double-stranded DNA and RNA molecules. HPLC of mixtures of DNA restriction fragments showed that fragments within the size range 0.125-23.1 kilobase were easily resolved. Supercoiled (form I) plasmid DNA molecules were readily separated from single-stranded circular DNA of the same length and from various DNA conformational isomers including nicked (form II) and linear (form III) species. Topological isomers generated from supercoiled plasmid DNA molecules by DNA topoisomerase I exhibited different retention times than supercoiled molecules. Supercoiled (form I) DNA molecules were resolved from fully relaxed (form IV) molecules. Synthetic oligonucleotides of 74 and 128 nucleotides in length were separated from failure sequences, as well as from other contaminating synthesis products. Single-stranded circular M13mp18 DNA molecules sufficiently pure for use in automated DNA sequencing systems were prepared by HPLC on a Gen-Pak FAX column. HPLC was also used to fractionate linear double-stranded porcine rotavirus genomic RNA fragments into size classes between 0.3 and 3 kilobase. Finally, HPLC of unfractionated Escherichia coli tRNA molecules resolved multiple species. In all cases, HPLC on Gen-Pak FAX was carried out in phosphate or Tris buffers at neutral pH in the presence of sodium chloride. Columns were not damaged by repeated exposure to impure samples, provided they were cleaned frequently with sodium hydroxide and acetic acid. Although procedures for resolution of the various size ranges for each class of DNA and RNA molecules require further optimization, our preliminary data on the separations obtained, the moderate salt concentrations employed, and the durability of the matrix suggest that this column merits further study.

A major single-stranded DNA binding protein from ovaries of the frog, Xenopus laevis, is lactate dehydrogenase

The most abundant single-stranded DNA binding protein (SSB) found in ovaries of the frog, Xenopus laevis, was purified to electrophoretic homogeneity. Under physiological conditions, the purified SSB lowered the Tm of poly[d(A-T)] and stimulated DNA synthesis by the homologous DNA polymerase DNA primase alpha complex on single-stranded DNA templates. These properties are characteristic of a bona fide single-stranded DNA binding protein. The Stokes radius of native SSB was calculated to be 45 A, corresponding to a molecular mass of about 140 kDa. On SDS polyacrylamide gels, the SSB migrated as a single band with a molecular mass of 36 kDa. We assumed, therefore, that the SSB was a tetramer of 36 kDa subunits. We subsequently discovered that the SSB was LDH, D-lactate dehydrogenase, EC 1.1.1.28. Purified SSB has high LDH specific activity. Following electrophoresis on SDS polyacrylamide gels, the 36 kDa subunits were renatured and exhibited LDH activity. The amino-acid composition of X. laevis SSB/LDH was similar to that of LDH from other species and to other reported single-stranded DNA binding proteins. Mammalian SSB/LDH also preferentially bound single-stranded DNA. Mammalian SSB/LDH bound to RNA as demonstrated by affinity chromatography on poly(A)-agarose and by its effect on translation of mRNA in vitro.