Electrophoretic analysis of EcoRI and HindIII restriction fragments of 2-;n supercoiled DNA of Saccharomyces cerevisiae indicated that this class of DNA is heterogeneous and probably consists of two types of molecules. Integration of the 2gm yeast DNA in E. coli plasmid pCR1 directly showed the existence of two types of molecules, as each of these could be individually inserted into separate bacterial plasmids. The difference between the two types of 2-jim circles is due to an inversion of about 1.6 X 106 daltons. The inversion is flanked by a reversed duplicated sequence of 0.45 X 106 daltons. Possible implications of this structure are discussed. Saccharomyces cerevisiae contains a class of closed circular duplex DNA molecules having a monomeric circumference of 2 ;1m and exhibiting the density of nuclear DNA (1, 2). This circular DNA, located outside the mitochondria (2) and nucleus (3), may be associated with a membrane fraction (4), and is here designated as episomal DNA. Analysis of the renaturation kinetics has shown that all 2-Am molecules have essentially the same base sequence without detectable repeated sequences (5).In this paper we report the integration of yeast episomal DNA in the Escherichta coli plasmid pCR1, which carries a gene conferring resistance to kanamycin. This has allowed us to separate and analyze two types of 2-,um DNA molecules that are identical in size and density, but produce different restriction fragments upon digestion with EcoRI or HindIII endonuclease. MATERIALS AND METHODSStrains and DNA Preparations. E. coli 490 (recA-, rkr, mk-) was obtained from G. Hobom. The pCR1 kanamycin resistance plasmid was isolated as closed circular DNA (6) from E. coli C600 transformed with this plasmid, after exposure of the cells to chloramphenicol (7). Yeast episomal DNA was isolated from S. cerevtsuie H1 as described (2) (Fig. lb, band 4). In electron micrographs this band was shown to consist of supercoiled DNA of about 4 Mm in length. Most preparations contained the minor bands 1 and 2 (Fig. lb), whose positions suggest that they are the relaxed.form of the 4-,um molecule and the supercoiled form of the 6-Am circular DNA class, respectively. Both 4-tam and 6-,um circles have been previously observed in episomal DNA (4,21,22).The migration rate of closed circular DNAs in 0.5% agarose plus ethidium bromide (100 ,g/ml) has been shown to be inversely proportional to the logarithm of the molecular weight (19). We have used this gel system to measure the molecular weight of supercoiled 2-gm DNA with pCRl DNA, phage PM2

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The Balbiani rings in the salivary gland polytene chromosomes of Chironomus tentans include the most active structural genes in this organ. Two of them (BR1 and BR2) contain repetitive sequences and are transcribed into giant RNA molecules. On Southern blots of restriction digests, we have identified fragments of genomic DNA which contain BR sequences. One of these fragments with a length of about 150 bp has been cloned and shown to hybridize preferentially to the BR1 transcription unit. Determination of its nucleotide sequence revealed several recognition sites for restriction enzymes which cleave the giant BR gene(s) into small pieces of approximately 240 bp. It is concluded that the cloned fragment represents part of the basic 240 bp repeat unit of a BR1 gene. Data obtained from partial restriction digests using the cloned DNA segment as a probe indicate that probably the entire BR1 gene comprises tandem repeats of 240 bp. Evidence is presented that the cloned BR1 sequence significantly cross-hybridizes to BR2 and to a lesser extent to BR6. BR2 sequences are present on a MboI fragment of 40 kb and seem to be organized in a very similar way as found for the BR1 gene.

The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for supertransformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-γ and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L−1 range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MFα1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha.

The early status of strain development for the production of interleukin (IL)-6, IL-8, IL-10, and interferon (IFN) gamma is described. The general approach to generating such strains was to amplify gene sequences encoding the mature forms of the various cytokines by PCR from commercially available cDNA sources. The design of the amplificates allowed an in-frame fusion to an MFalpha1 leader segment contained in two basic expression vectors, pFPMT121-MFalpha1 and pTPSMT-MFalpha1. The two vectors differ in that one harbors the methanol-inducible FMD promoter and the other the constitutive TPS1 promoter as control elements for heterologous gene expression. The most advanced process development example is that of IFNalpha-2a. Here, the MOX promoter derived from another key gene of methanol metabolism is used for expression control. The successful development of a production process for Hansenula polymorpha-derived IFNalpha-2a is summarized. This was achieved by combining genetic engineering of suitable production strains with improved processing capabilities for the secreted cytokine, and by purification procedures from cultures grown in yeast extract-peptone-glycerol-based media.