Cell wall inulinase (EC 3.2. I .7) was pirified from K/ywvwt.ws r~~arshr~s var. mtrsicms (formerly K. Jrugilis) and its N-terminal 33-amino acid sequence was established. PCR amplification of cDNA with 2 sets of degenerate primers yielded a gcnomic probe which was then used to screen a genomic library established in the YEp351 yeast shuttle vector. One of the selected recombinant plasmids allowed an invertasc-negative S~~clrcrrurr~~~s cerevisicre mutan to grow on inulin. It was shown to contain an inulinase gene (INU I) encoding a 555-amino acid precursor protein with a typical N-terminal signal pcptide. The sequence of inulinase displays a high similarity (67%) IO S. ceruisictr invertasc. suggesting a common evolutionary origin for yeas1 p-fructosidases with different substrate preferences.

The Mig1 repressor is a key effector in glucose repression in the yeast Saccharomyces cerevisiae. To gain further insights into structure-function relationships, we have now cloned the MIG1 homologue from the yeast Kluyveromyces marxianus. The amino acid sequence deduced from KmMIG1 differs significantly from ScMig1p outside the highly conserved zinc fingers. However, 12 discrete conserved motifs could be identified in a multiple alignment that also included the K. lactis Mig1p sequence. We further found that KmMig1p is fully functional when expressed in S. cerevisiae. First, it represses the SUC2 promoter almost as well as ScMig1p. This repression requires the Cyc8 and Tup1 proteins and is dependent on a C-terminal region comprising several conserved leucine-proline repeats. Second, KmMig1p is regulated by glucose in S. cerevisiae, and a KmMig1-VP16 hybrid activator is inhibited by the ScSnf1p kinase in the absence of glucose. This suggests that KmMig1p has retained the ability to interact with several S. cerevisiae proteins, and reinforces the notion that the conserved motifs are functionally important. Finally, we found that the physiological role of Mig1p also is conserved in K. marxianus, since KmMig1p represses INU1, the counterpart of SUC2 in this organism.

Sequence comparisons between [9,10]. Moreover, in addition to its galactokinase activity, the K. lactis GALl gene product has a regulatory function required for the induction pathway, upstream of K1LAC9 [11]. Interestingly, a potential MIGl-binding site is found in the GALl promoter of K. lactis suggesting the involvement of a Migl-like protein in glucose repression of galactose-lactose catabolism in K. lactis.The SUC2 gene encoding invertase in S. cerevisiae, on the other hand, displays two MIG 1-binding sites in its promoter [3] and two putative MIG1 sites are also found [12] upstream of the INU1 gene encoding inulinase (a closely related enzyme) in Kluyveromyces marxianus [13], suggesting that the latter gene also may be repressed by a Migl homologue.In this paper, we report the cloning and sequencing of the K. lactis MIG1 gene and we show that the encoded protein is functional in S. cerevisiae.

HighlightsDeep sequencing has potential as an improved adventitious virus screening method.15 laboratories sequenced a common reagent containing 25 target viruses.6 viruses were detected by all lab, the remainder were detected by 4–14 labs.A wide range of sample preparation and bioinformatics methods is currently used.A common reference material is essential to enable results to be compared.

The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses makes it a powerful tool for broad microbial investigations, such as evaluation of novel cell substrates that may be used for the development of new biological products. However, like any new assay, regulatory applications of HTS need method standardization. Therefore, our three laboratories initiated a study to evaluate performance of HTS for potential detection of viral adventitious agents by spiking model viruses in different cellular matrices to mimic putative materials for manufacturing of biologics. Four model viruses were selected based upon different physical and biochemical properties and commercial availability: human respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), feline leukemia virus (FeLV), and human reovirus (REO). Additionally, porcine circovirus (PCV) was tested by one laboratory. Independent samples were prepared for HTS by spiking intact viruses or extracted viral nucleic acids, singly or mixed, into different HeLa cell matrices (resuspended whole cells, cell lysate, or total cellular RNA). Data were obtained using different sequencing platforms (Roche 454, Illumina HiSeq1500 or HiSeq2500). Bioinformatic analyses were performed independently by each laboratory using available tools, pipelines, and databases. The results showed that comparable virus detection was obtained in the three laboratories regardless of sample processing, library preparation, sequencing platform, and bioinformatic analysis: between 0.1 and 3 viral genome copies per cell were detected for all of the model viruses used. This study highlights the potential for using HTS for sensitive detection of adventitious viruses in complex biological samples containing cellular background. Recent high-throughput sequencing (HTS) investigations have resulted in unexpected discoveries of known and novel viruses in a variety of sample types, including research materials, clinical materials, and biological products. Therefore, HTS can be a powerful tool for supplementing current methods for demonstrating the absence of adventitious or unwanted viruses in biological products, particularly when using a new cell line. However, HTS is a complex technology with different platforms, which needs standardization for evaluation of biologics. This collaborative study was undertaken to investigate detection of different virus types using two different HTS platforms. The results of the independently performed studies demonstrated a similar sensitivity of virus detection, regardless of the different sample preparation and processing procedures and bioinformatic analyses done in the three laboratories. Comparable HTS detection of different virus types supports future development of reference virus materials for standardization and validation of different HTS platforms.