Harm van Heerikhuizen

High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression

Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with a high mitotic stability under non-selective conditions. Since high stability can best be assured by integration of the vector into chromosomal DNA we have set out to design a vector that is able to integrate into the yeast genome in a large number of copies. The rDNA locus appeared to be an attractive target for such multiple integration since it encompasses 100-200 tandemly repeated units. Plasmids containing several kb of rDNA for targeted homologous recombination, as well as the deficient LEU2-d selection marker were constructed and, after transformation into yeast, tested for both copy number and stability. One of these plasmids, designated pMIRY2 (for multiple integration into ribosomal DNA in yeast), was found to be present in 100-200 copies per cell by restriction analysis. The pMIRY2 transformants retained 80-100% of the plasmid copies over a period of 70 generations of growth in batch culture under non-selective conditions. To explore the potential of pMIRY2 as an expression vector we have inserted the homologous genes for phosphoglycerate kinase (PGK) and Mn2+-dependent superoxide dismutase (SOD) as well as the heterologous genes for thaumatin from Thaumatococcus danielli (under the GAPDH promoter), into this plasmid and analyzed the yield of the various proteins. Under optimized conditions the level of PGK in cells transformed with pMIRY2-PGK was about 50% of total soluble protein. The yield of thaumatin in the pMIRY2-thaumatin transformants exceeded by about a factor of 100 the level of thaumatin observed in transformants carrying only a single thaumatin gene integrated at the TRP1 locus in chromosome IV.

Monitoring macromolecular complexes involved in the chaperonin-assisted protein folding cycle by mass spectrometry

We have used native mass spectrometry to analyze macromolecular complexes involved in the chaperonin-assisted refolding of gp23, the major capsid protein of bacteriophage T4. Adapting the instrumental methods allowed us to monitor all intermediate complexes involved in the chaperonin folding cycle. We found that GroEL can bind up to two unfolded gp23 substrate molecules. Notably, when GroEL is in complex with the cochaperonin gp31, it binds exclusively one gp23. We also demonstrated that the folding and assembly of gp23 into 336-kDa hexamers by GroEL-gp31 can be monitored directly by electrospray ionization mass spectrometry (ESI-MS). These data reinforce the great potential of ESI-MS as a technique to investigate structure-function relationships of protein assemblies in general and the chaperonin-protein folding machinery in particular. A major advantage of native mass spectrometry is that, given sufficient resolution, it allows the analysis at the picomole level of sensitivity of heterogeneous protein complexes with molecular masses up to several million daltons.

How does lysozyme penetrate through the bacterial outer membrane

Lysozyme fails to penetrate through the outer membrane of stationary phase cells of Escherichia coli when it is simply added to suspensions of plasmolyzed cells. Lysozyme penetrates the outer membrane only when these cells are exposed to a mild osmotic shock in the presence of EDTA and lysozyme. In the presence of Mg2+, the outer membrane is stabilized sufficiently so that there is no lysozyme penetration during osmotic shock. If Mg2+ is added after an osmotic shock has been used to cause lysozyme to penetrate a destabilized outer membrane, the outer membrane is stabilized once again. In this case however, cells are converted to spheroplasts by the lysozyme which has gained access to the murein layer prior to the addition of Mg2+. Mg2+ stabilizes the outer membranes of these spheroplasts sufficiently so that they remain immune to lysis even in the absence of osmotic stabilizers such as sucrose. These results are discussed in terms of current information on the structure of the murein layer and the outer membrane.

Molecular cloning of the rDNA of Saccharomyces rosei and comparison of its transcription initiation region with that of Saccharomyces carlsbergensis

We have cloned one complete repeating unit of rDNA from Saccharomyces rosei and determined its physical and genetic organization. Heteroduplex analysis of the rDNA units from S. rosei and S. carlsbergensis shows that the nontranscribed spacers are largely nonhomologous in sequence, whereas the transcribed regions are essentially homologous. We also determined the transcription initiation site for the 37S precursor RNA on S. rosei rDNA. Sequence comparison of the region surrounding the site of transcription initiation for the 37S RNA with the corresponding region of S. carlsbergensis revealed extensive homology from position -9 downstream into the external transcribed spacer. Very little homology was observed between position -9 and -55, but some homologous tracts are present upstream from position -55.

Heterogeneity in the ribosomal RNA genes of the yeast Yarrowia lipolytica; cloning and analysis of two size classes of repeats

Southern blotting of DNA from the ascomycetous yeast Yarrowia lipolytica revealed two major size classes of DNA units coding for rRNAs, which differ in length by about 1000 bp. We have cloned an rDNA unit of each size class. R-looping experiments revealed that the rRNA genes of both units are uninterrupted; subsequent heteroduplex analysis showed that the size difference both units is located within the nontranscribed spacer. Sequence analysis revealed that a major part of these spacers consists of a complex pattern of repetitions in periodicities of up to about 150 bp and that the difference between both rDNA units are located mainly in this repetitive region. Apart from different lengths of the repetitive regions, both rDNA units also reveal extended microheterogeneity within their homologous parts. Furthermore, no gene for 5S rRNA was observed in the spacer region. Therefore, the organization of the spacer of Yarrowia rDNA is clearly different from that of Saccharomyces cerevisiae.

Regulation of expression of the amino acid transporter gene BAP3 in Saccharomyces cerevisiae.

The BAP3 gene of Saccharomyces cerevisiae encodes a protein with a high similarity to the BAP2 gene product, a high‐affinity permease for branched‐chain amino acids. In this paper, we show that, like BAP2, the expression of the BAP3 gene in S. cerevisiae is induced by the addition of branched‐chain amino acids to the medium. Unexpectedly, most other naturally occurring L‐amino acids found in proteins (with the exception of proline, lysine, arginine and histidine) have the same effect on the expression of BAP3. The induction of BAP3 expression appears to be dependent on Stp1p, a nuclear protein, previously shown to be involved in pre‐tRNA maturation and also required for the expression of BAP2, as induction is no longer observed in an stp1 − mutant. The transcriptional regulator Leu3p is not involved in the induction of BAP3 expression, but may act as a repressor of BAP3 expression in the absence of leucine, as can be inferred from a transcriptional analysis in a Δleu3 mutant. By extensive deletion analysis of the BAP3 promoter fused to a GUS reporter, as well as by fusions of different parts of the BAP3 promoter to a LacZ reporter, we have found that a portion of the BAP3 promoter from − 418 to − 392 relative to the ATG start codon is both necessary and sufficient for the Stp1p‐dependent induction of BAP3 expression by (most) amino acids. We have therefore named this sequence UASaa (amino acid‐dependent upstream activator sequence). Neither Stp1p nor Leu3p appear to bind to the UASaa, at least in vitro, as judged from gel retardation assays. Sequences similar to the UASaa can be found in the promoters of BAP2, PTR2 and TAT1 ; genes that, like BAP3, encode permeases inducible by amino acids, suggesting that amino acid induction of all these genes is exerted via a common mechanism.

Evolution of yeast ribosomal DNA: Molecular cloning of the rDNA units of Kluyveromyces lactis and Hansenula wingei and their comparison with the rDNA units of other Saccharomycetoideae

SummaryWe have studied the evolution of the yeast ribosomal DNA unit to search for regions outside the rRNA genes that exhibit evolutionary constraints and therefore might be involved in control of ribosome biosynthesis. We have cloned one complete rDNA unit of Kluyveromyces lactis and Hansenula wingei and established the physical and genetic organisation of both units. Both species belong to the subfamily of the Saccharomycetoidea. The lengths of the rDNA units of K. lactis and H. wingei are 8.6 and 11.1 kb respectively, and both comprise the 5S rRNA gene in addition to the large rRNA operon. Sequence conservation was monitored by restriction enzyme mapping as well as heteroduplex analysis of the two cloned rDNA units with S. carlsbergensis rDNA. These analyses showed that, phylogenetically, K. lactis is closer to S. carlsbergensis than H. wingei. The non-transcribed spacers (NTS) of both K. lactis and H. wingei have diverged completely from S. carlsbergensis; moreover in H. wingei the NTS are about double the length of these in the other two species. The transcribed spacers of both K. lactis and H. wingei contain conserved tracts. A homologous sequence of about 60 bp was found in the middle of the external transcribed spacer of H. wingei upon heteroduplexing with S. carlsbergensis rDNA, whereas the sequence at the transcription initiation site itself was insufficiently homologous to form a duplex. The sequence of the homologous region was determined both in H. wingei and K. lactis and compared with that of S. carlsbergensis. The function of this conserved element within the external transcribed spacer is discussed.

Heterogeneity in the ribosomal family of the yeast Yarrowia lipolytica: genomic organization and segregation studies

The cloned r-DNA units of Yarrowia lipolytica [Van Heerikhuizen et al., 39 (1985) 213-222] and their restriction fragments have been used to probe blots of genomic DNA of this yeast. Wild-type and laboratory strains were shown to contain two-to-five types of repeated units, each strain displaying a specific pattern. By comparing their restriction patterns, we could localize the differences between units within their spacer region. Tetrad analysis strongly suggested a clustered organization of each type of repeat as well as the occurrence of meiotic exchanges within the r-DNA family. Chromosome loss was induced by benomyl and allowed to map several r-DNA clusters on the same chromosome. All those results indicate that the Y. lipolytica r-DNA gene family is quite different from other yeasts.

Characterization of a cDNA clone encoding molluscan insulin-related peptide V of Lymnaea stagnalis

A cDNA clone encoding molluscan insulin-related peptide V (MIP V) was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis, using a heterologous screening with a previously identified MIP II cDNA. The MIP V cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, however, in this case connected by two distinct C peptide, C alpha and C beta, instead of one single C peptide. This phenomenon, which is shared by the MIP II precursor, represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIPs V, I and II, differ remarkably in primary structure; in contrast, the C alpha peptide domains are almost identical. MIP V has only limited sequence similarity with insulins and related peptides. Both MIP V and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I, II and V genes are expressed in a single type of neuron: the growth controlling neuroendocrine light green cells of the Lymnaea CNS.

Molecular cloning of G protein α subunits from the central nervous system of the mollusc Lymnaea stagnalis

The central nervous system of the pond snail, Lymnaea stagnalis, contains many large, identified neurons which can be easily manipulated making it an advantageous model system to elucidate in vivo the architecture of neuronal signal transduction pathways. We have isolated three cDNA clones encoding G protein α subunits that are expressed in the Lymnaea CNS, i.e. Gαo, Gα, and Gαi. The deduced proteins exhibit a very high degree of sequence identity to their vertebrate and invertebrate counterparts. The strong conservation or G protein α subunits suggests that functional insights into G protein‐mediated signalling routes obtained through the experimental amenability of the Lymnaea CNS will have relevance for similar pathways in the mammalian brain.