Stefan J. Surzycki

Factors effecting expression of vaccines in microalgae

PhycoBiologics is developing an oral vaccine delivery system using vaccines expressed in the chloroplast of microalgae. Despite many advances in plastid transformation technology, levels of expression remain inconsistent. We have concluded that the main factors affecting the level of recombinant protein expression in the chloroplast of Chlamydomonas are: codon optimization, protease activity, protein toxicity and transformation-associated genotypic modification.

Both the chloroplast and nuclear genomes of Chlamydomonas reinhardi share homology with Escherichia coli genes for transcriptional and translational components

SummaryConsiderable DNA sequence homology can be detected between the Escherichia coli genes coding for translational and transcriptional components and both the chloroplast and nuclear genomes of Chlamydomonas reinhardi. Labeled chloroplast DNA was demonstrated to hybridize to DNA fragments of the transducing phages λfus3 and λspc2 that encode ribosomal proteins of the α and S10 operons. Further, chloroplast DNA probes hybridize to fragments of λrtfd 18 that encode the β and β′ subunits of RNA polymerase. The regions homologous to the ribosomal protein and RNA polymerase genes were located on the chloroplast DNA physical map by probing restriction fragments of chloroplast DNA with phage or plasmid fragments carrying these E. coli genes. Probing nuclear DNA with bacterial gene probes revealed DNA fragments homologous to elongation factor and ribosomal protein genes. Most surprisingly, sequences homologous to the β subunit of RNA polymerase were found not only in chloroplast DNA but in nuclear DNA as well.

Chloroplast gene expression in Chlamydomonas reinhardi

SummaryThe transcriptional activities of the chloroplast genome of Chlamydomonas reinhardi during the synchronous cell cycle and at various stages of the greening process have been characterized by hybridization of pulse labeled RNA to separated fragments of chloroplast DNA digested with EcoRI endonuclease. The results demonstrate that the EcoRI-chloroplast DNA fragments can be divided into three classes with respect to gene expression: (1) fragments in which gene expression occurs stage specifically, (2) fragments in which gene expression occurs continously, and (3) fragments in which gene expression is absent. A typical example of class 1 DNA fragments are those which contain chloroplast rRNA genes. Several other fragments containing mRNA species are also transcribed stage specifically. The DNA fragment containing the mRNA gene for the large subunit of RuBP-carboxylase appears to belong to class 2. Three EcoRI fragments (19, 20, 22) are not transcribed at all. These fragments were not expressed even when the chloroplast DNA was transcribed in vitro by E. coli RNA polymerase. The buoyant density analysis of DNA fragments shows a distinct base composition hetereogeneity. The three silent fragments have an extremely low bouyant density and are localized in a cluster on the physical map of Rochaix (1978). Four high density regions can be recognized on the map, two of which involve genes for chloroplast rRNAs and one for mRNA for the large RuBP-carboxylase subunit.

Organization and structure of plastome psbF, psbL, petG and ORF712 genes in Chlamydomonas reinhardtii

SummaryWe have determined the nucleotide sequence of a 5159 base-pair (bp) region of the Chlamydomonas reinhardtii plastome containing three photoelectron transport genes, psbF, psbL and petG, and an unusual open reading frame, ORF712. The photosynthetic genes have an unprecedented arrangement. psbF and psbL are located in close proximity to petG, and are not grouped with two other genes of the cytochrome b559 locus, psbE and ORF42. ORF712, located adjacent to psbL, has homology at its 5′-and 3′-ends to the ribosomal protein rps3 gene, but contains, a central 437 residue domain that lacks similarity to any other known sequence. These sequences add to the growing body of evidence that the chloroplast genome of C. reinhardtii has a significantly different gene arrangement to its counterpart in plants. The structure of ORF712 also provides another example of a phenomenon we have discovered with C. reinhardtii RNA polymerase genes (Fong and Surzycki 1992); namely, that the algal plastome contains chimeric genes in which reading frames with homology to known genes are juxtaposed in-frame with long coding regions of unknown identity.

Chloroplast and nuclear genomes of Chlamydomonas reinhardtii share homology with Escherichia coli genes for DNA replication, repair and transcription

SummaryMost of the cpDNA genes studied to date are genes encoding elements of the photosynthetic apparatus and translational machinery. Much less is known about genes encoding the polypeptides involved in transcription, cpDNA replication, recombination and repair. The similarities between bacterial and some cpDNA genes were exploited to identify some of these chloroplast genes using bacterial probes. Probes derived from the Escherichia coli genes dnaA, recA, uvrC transcriptional factor rho, and rpoC were used to search for homologous DNA sequences in chloroplst and nuclear genomes of Chlamydomonas reinhardtii. Regions homologous to all of these genes were located on the cpDNA physical map by probing restriction fragments of cpDNA with plasmid fragments containing these genes. Probing nuclear DNA with bacterial gene probes revealed DNA fragments homologous to dnaA and rpoC genes.

E. coli ribosomal proteins are cross reactive with antibody prepared against Chlamydomonas reinhardi chloroplast ribosomal subunit.

SummaryAntisera prepared against purified Chlamydomonas reinhardi small chloroplast ribosomal subunit, judged homogenous by sucrose gradient velocity sedimentation and RNA gel electrophoresis was immunologically cross reactive with E. coli ribosomal proteins. The results of three different experimental approaches, namely Ouchterlony double diffusion, sucrose gradient velocity sedimentation and two dimensional crossed immunoelectrophoresis indicate that both E. coli ribosomal subunits and the chloroplast large ribosomal subunit contain proteins which show antigenic similarity to the chloroplast small ribosomal subunit proteins. However, cytoplasmic ribosomal subunits did not contain proteins which were cross reactive with immune antisera.

Location of Binding Sites for RNA Polymerase II from Wheat Germ and from Human Placenta on Adenovirus 2 DNA

SummaryElectron microscopic visualization of binary complexes between eukaryotic RNA polymerases and Adenovirus 2 (Ad 2) DNA was used to locate specific binding sites for the enzymes. RNA polymerase II from human placenta binds to 10–16 distinct sites depending on the ratio of enzyme to DNA and the divalent cation present in the binding mixture. Wheat germ RNA polymerase binds to 12–14 strong binding sites and 2–3 weaker sites, all but one of which correspond to binding sites for the placental enzyme. At least six of the strong binding sites for both enzymes correspond to promoters known to be active in vivo.As a test of the two-state model for transcription initiation, we examined binding of wheat germ RNA polymerase II to Ad 2 DNA at 0° and 37°. The extent of binding was the same at the two temperatures and the distributions of binary complexes were virtually identical. This observation, in conjunction with results presented previously, is strong support for the existence of I and RS complexes in eukaryotic systems.

Screening recombinant clones containing sequences homologous to Escherichia coli genes using single-stranded bacteriophage vector

Detection and isolation of Escherichia coli clones carrying vectors with foreign DNA sequences partially homologous to specific E. coli genes is difficult because denatured DNA in the host genome can hybridize with the probe. In this paper we present a procedure which simplifies this task by using bacteriophage M13 as the cloning vector. The procedure takes advantage of the secretory properties of the phage, as well as the property of nitrocellulose membrane to bind protein and single-stranded DNA but not double-stranded DNA. This procedure is shown to be effective in identifying E. coli clones containing sequences of Chlamydomonas reinhardtii chloroplast DNA that are homologous to the rpoC gene of E. coli. We suggest that this procedure can be used generally for rapid isolation of DNA sequences that are homologous to E. coli genes.

Transcription of adenovirus 2 DNA by human RNA polymerase II in vitro

SummaryThe interaction of Adenovirus 2 DNA and human placental RNA polymerase II in vitro satisfies criteria that suggest that at least some fraction of our purified polymerase preparations corresponds to prokaryotic holoenzyme and is able to initiate transcription at “true” promoters: (1) The purified enzyme forms highly stable complexes at specific sites on Ad 2 DNA; Kass=1–2x1012 M-1. (2) Transcription of Ad 2 DNA from pre-formed complexes with human RNA polymerase II is resistant to poly I. (3) Many of the stable-binding sites correspond to Ad 2 promoters known to be active in vivo.We also present evidence consistent with a twostate (I and RS) model (Chamberlin et al. 1976; Travers 1974) for the interaction of human RNA polymerase II with Ad 2 DNA. These experiments, which are similar to those described previously in studies of wheat germ RNA polymerase II (Seidman et al. 1979), indicate that the mechanisms of transcription initiation and promoter site selection in eukaryotic and prokaryotic systems may be very similar.

Inactivation of E. coli RNA polymerase by polyriboinosinic acid: heterogeneity of RS complexes.

SummaryPolyriboinosinic acid (poly I) inhibits initiation of transcription by binary complexes formed between Adenovirus 2 DNA and E. coli RNA polymerase holozyme. In the presence of poly I, just as in the presence of rifampicin, initiation of transcription exhibits a sigmoidal dependence on the temperature at which the binary complexes are formed. This indicates that I (closed) complexes between Ad 2 DNA and RNA polymerase are rapidly inactivated by poly I, but that RS (open) complexes are relatively resistant. However, even among the RS complexes, at least two classes can be distinguished on the basis of the degree to which they are resistant to poly I: RS-1 complexes are somewhat sensitive to poly I (half-time of inactivation approximately 10 min) while RS-2 complexes are almost completely resistant to the inhibitor (half-time of inactivation approximately 10 h). For both types of RS complex, the degree of sensitivity to poly I is ionic strength-dependent.