A laboratory heterodyne emission spectrometer at submillimeter wavelengths.

We present first results on a newly built broadband emission spectrometer for the laboratory making use of a double sideband (DSB) heterodyne receiver. The new spectrometer is perfectly suited for high-resolution emission spectroscopy of molecules of astrophysical importance. The current SIS receiver operates at RF frequencies between 270 and 390 GHz, coincident with Band 7 of the ALMA telescope. The instantaneous bandwidth is 5 GHz (DSB). In this work the full spectrometer and its components are described. Its performance, in particular its sensitivity, stability, reproducibility and systematic errors, is characterized in detail. For this purpose very broad band emission spectra of methyl cyanide have been recorded and compared to theoretical spectra. Isotopic variants are found in natural abundance and features attributed to vibrationally excited species are all recorded in the same spectrum. The performance of the new spectrometer is compared extensively to that of a traditional FM-absorption spectrometer and to recent versions of chirped-pulse spectrometers operated in the mm-wave regime. Further applications and future advancements of the current instrument are discussed.

Overview of protein expression in Pichia pastoris.

Pichia pastoris is a methylotrophic yeast and can be used as a heterologous expression system. This microorganism is as easy to manipulate as Escherichia coli, but has many of the advantages of eukaryotic expression (e.g., protein processing, folding, and post-translational modifications), and it is faster, easier, and cheaper to use than other eukaryotic expression systems, such as baculovirus or mammalian tissue culture. It also generally yields higher expression levels. This overview discusses important considerations for the use of Pichia pastoris, including strains for expression, expression plasmids, transformation by integration, and post-translational modifications. Examples of expression are given and finally, legal issues regarding patent rights for heterologous protein expression in Pichia pastoris are described.

Recombinant protein expression in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.

Production of a recombinant bovine enterokinase catalytic subunit in the methylotrophic yeast Pichia pastoris.

We describe the heterologous expression of a 26.3 kD protein containing the catalytic domain of bovine enterokinase (EKL) in the methylotrophic yeast Pichia pastoris. A highly active protein is secreted and glycosylated, and it has the native amino-terminus of EKL. The cDNA encoding EKL was cloned with the KEX2 protease cleavage site following the alpha mating factor prepro secretion signal from Saccharomyces cerevisiae. The secreted EKL was easily purified from the few native proteins found in the P. pastoris fermentation supernatant, using ion exchange and affinity chromatography. The yield of the purified EKL was 6.3 mg per liter of fermentation culture. This is significantly higher than previous reports of expressions in E. coli and COS cells. The ability of this highly specific protease to cleave immediately after the carboxyl-terminal residue of the (Asp)4-Lys recognition sequence allows regeneration of native amino-terminal residues of recombinant proteins. Its application is demonstrated by the removal of thioredoxin (TrxA), and polyhistidine fusion partners from proteins of interest.

Silicon-responsive cDNA clones isolated from the marine diatom Cylindrotheca fusiformis.

In organisms ranging from single-celled algae to mammals, including humans, silicon is essential for, and actively participates in, a variety of life processes. It has become clear that silicon (i) acts as a metabolite affecting a variety of cellular processes, and (ii) regulates gene expression. However, the mechanisms by which silicon (i.e., Na2SiO3.9H2O, in the present study) acts are not clear, due to inherent methodological difficulties. As part of our program to understand how silicon acts in biological systems, we present the first isolation of cDNA clones derived from silicon-responsive mRNAs, from the marine diatom Cylindrotheca fusiformis. We distinguish between clones responding only to silicon starvation and replenishment, and those also responding to other cellular conditions. Some of the clones can be identified by similarity to other genes, and should be useful as probes to isolate genes from other organisms. Isolation of these clones provides the means to (i) identify metabolic pathways affected by silicon, and (ii) investigate the mechanism(s) of silicon-regulated gene expression.

Electroporation-stimulated recombination in yeast.

Saccharomyces cerevisiae cells treated by high voltage and made transformation-competent (electroporation) are also made hyper-recombinational as determined by an assay that measures interchromosomal mitotic recombination between chromosome III homologs, each containing mutant heteroallelic copies of the trp1 and his3 genes. There is a 10-fold stimulation of Trp+ and 21-fold stimulation of His+ prototrophs. Although this stimulation coincides with conditions for maximal transformation competence it is independent of the presence of transforming plasmid DNA. Electroporation does not increase the reversion frequency of these mutations, nor is there a stimulation in Ty transposition. Among the electroporation-stimulated Trp+ and His+ recombinants there is no dramatic difference in the pattern of events: that is to say that, while there is an increase in the number of recombinants, the distribution of gene conversion and cross-over events among the stimulated recombinants is not significantly altered compared to spontaneously arising Trp+ and His+ recombinants. This electroporation-stimulated recombination is abolished in an isogenic rad52 mutant strain consistent with the increase in Trp+ and His+ prototrophs being the result of a stimulation of a RAD52-dependent recombination pathway.

A novel recombinator in yeast based on gene II protein from bacteriophage f1.

Interchromosomal mitotic recombination in yeast can be stimulated by the protein encoded by gene II of bacteriophage f1. The normal role of the gene II enzyme is to make a site-specific cleavage of a particular strand of the duplex form of the bacteriophage DNA at the origin of DNA replication. The gene II protein was expressed in yeast in an attempt to determine the role of nicked DNA in the initiation of recombination. Stimulation of recombination in yeast by the gene II protein was dependent on the presence of a recognition site for gene II enzyme in the region being assayed. Recombination was stimulated in both directions from the gene II recognition site but showed a directional bias. The distribution of alleles among the recombinants indicated that the chromosome with the gene II recognition site acted as the recipient in gene conversion events.

Analysis of interchromosomal mitotic recombination.

A novel synthetic locus is described that provides a simple assay system for characterizing mitotic recombinants. The locus consists of the TRP1 and HIS3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Defined trp1 and his3 alleles have been generated that allow the selection of interchromosomal recombinants in this interval. Trp+ or His+ recombinants can be divided into several classes based on coupling of the other alleles in the interval. The tight linkage of the CRY1 and MAT loci, combined with the drug resistance and cell type phenotypes that they respectively control, facilitates the classification of the recombinants without resorting to tetrad dissection. We present the distribution of spontaneous recombinants among the classes defined by this analysis. The data suggest that the recombination intermediate can have regions of symmetric strand exchange and that co-conversion tracts can extend over 1-3 kb. Continuous conversion tracts are favored over discontinuous tracts. The distribution among the classes defined by this analysis is altered in recombinants induced by UV irradiation.

Mechanism for dynamic changes in stenotic severity.

The mechanism responsible for rapid changes in stenotic severity or resistance due to alterations in perfusion pressure and distal resistance is addressed by this study. An in vitro, eccentric arterial stenosis model was created using 15 canine carotid arteries cannulated with silicone plugs containing special pressure-transducing catheters designed to measure pressure directly, within the stenosis. The vessels were perfused at perfusion pressures of 150, 100, and 75 mmHg and at two levels of distal resistance while perfusion pressure, distal pressure, stenotic pressure, and flow were recorded. Orthogonal arteriograms were performed. Stenotic resistance was calculated as (perfusion pressure--distal pressure)/flow. All variables changed significantly (P less than 0.05) with decreases in perfusion pressure. Stenotic resistance always increased (P less than 0.02) as perfusion pressure or distal resistance decreased. These dynamic changes in stenotic resistance occurred at stenotic pressures well above the atmospheric, extraluminal pressure. Arteriographic data demonstrated decreasing stenotic luminal area with decreasing stenotic pressure. These results confirm the assertion that rapid changes in stenotic resistance are related to changes in stenotic luminal area and are not due to extrinsic forces. The principles of the Starling resistor are, therefore, not applicable to dynamic arterial stenoses. This information is immediately relevant to clinical situations in which complaint, severe stenosis results in ischemia.

The nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae: a potential adenine nucleotide binding amino acid sequence and a nonessential acidic carboxyl terminal region.

The RAD3 gene of Saccharomyces cerevisiae is required for excision of pyrimidine dimers and is essential for viability. We present the nucleotide sequence of the RAD3 protein coding region and its flanking regions, and the deduced primary structure of the RAD3 protein. In addition, we have mapped the 5' end of RAD3 mRNA. The predicted RAD3 protein contains 778 amino acids with a calculated molecular weight of 89,779. A segment of the RAD3 protein shares homology with several adenine nucleotide binding proteins, suggesting that RAD3 protein may react with ATP. The twenty carboxyl terminal amino acids of RAD3 protein are predominantly acidic; however, deletion of this acidic region has no obvious effect on viability or DNA repair.

Expression of the RAD1 and RAD3 genes of Saccharomyces cerevisiae is not affected by DNA damage or during the cell division cycle.

The RAD1 and RAD3 genes of Saccharomyces cerevisiae are required for excision repair of UV damaged DNA. In addition, the RAD3 gene is essential since rad3 deletions are recessive lethals. We have examined the induction of the RAD1 and RAD3 genes by DNA damage and during the cell division cycle. We have made fusions of the RAD1 and RAD3 genes with the Escherichia coli lacZ gene encoding beta-galactosidase. Beta-galactosidase activity was measured in a Rad+ yeast strain containing the RAD1-lacZ or the RAD3-lacZ fusion, either in a multicopy replicating plasmid or as a single copy integrant resulting from transformation with an integrating plasmid which transforms yeast by homologous recombination in the yeast genome. No induction of beta-galactosidase activity occurred after ultraviolet light (UV) or 4-nitroquinoline-1-oxide (NQO) treatment. Haploid cells of mating type a were synchronized by treatment with alpha factor and beta-galactosidase activity was determined during different cell cycle stages. No change in beta-galactosidase activity was observed in the strain containing the RAD1-lacZ or the RAD3-lacZ fusion integrated in the yeast genome.

Isolation and characterization of the RAD2 gene of Saccharomyces cerevisiae.

We have cloned the RAD2 gene of Saccharomyces cerevisiae and used it to determine the size and direction of its transcript and to make rad2 deletion mutants. The RAD2 gene encodes a 3.3-kb transcript and the direction of transcription is leftwards, from EcoRI towards Bg/II. Deletions of the RAD2 gene have no effect on viability of vegetative cells or spores, or on sporulation.

Molecular cloning and characterization of the RAD1 gene of Saccharomyces cerevisiae.

We have cloned the RAD1 gene of Saccharomyces cerevisiae and physically mapped it to a 4.0-kb DNA fragment from chromosome XVI. The RAD1 gene determines a transcript of 3.1 kb, and the direction of transcription was found to be leftwards, from EcoRI towards Bg/II (Fig. 1). Deletions of the RAD1 gene were made and were found to have no effect on viability of vegetative cells or spores, or on sporulation.

Isolation and characterization of the RAD3 gene of Saccharomyces cerevisiae and inviability of rad3 deletion mutants.

The RAD3 gene of Saccharomyces cerevisiae is required for nicking of DNA containing pyrimidine dimers or interstrand crosslinks. We have cloned the RAD3 gene and physically mapped it to 2.6 kilobase of DNA. A DNA segment of the cloned RAD3 insert was ligated into plasmid YIp5, which transforms yeast by homologous integration, and shown to integrate at the RAD3 site in chromosome V, thus verifying the cloned DNA segment to be the RAD3 gene and not a suppressor. The RAD3 gene encodes a 2.5-kilobase mRNA, extending between the Kpn I site and the Sau3A1/BamHI fusion junction in plasmid pSP10, and the direction of transcription has been determined. The 2.5-kilobase transcript could encode a protein of about 90,000 daltons. We also show the deletions of the RAD3 gene to be recessive lethals, indicating that the RAD3 gene plays an important role in other cellular processes in addition to incision of damaged DNA.