Regulation of acid phosphatases in anAspergillus niger pacC disruption strain

AnAspergillus niger strain has been constructed in which the pH-dependent regulatory gene,pacC, was disrupted. ThepacC gene ofA. niger, like that ofA. nidulans, is involved in the regulation of acid phosphatase expression. Disruptants were identified by a reduction in acid phosphatase staining of colonies. Southern analysis demonstrated integration of the disruption plasmid at thepacC locus and Northern analysis showed that the disruption strain produced a truncatedpacC mRNA of 2.2 kb (as compared to 2.8 kb in the wild type). The strain carrying thepacC disruption was used to assign thepacC gene to linkage group IV; this was confirmed by CHEF electrophoresis and Southern analysis. This strain further allowed us to determine which extracellular enzyme and transport systems are under the control ofpacC inA. niger. Expression of theA. niger pacC wild-type gene and the truncatedpacC gene showed that, in contrast to the auto-regulated wild-type expression, which was elevated only at alkaline pH, the truncatedpacC gene was deregulated, as high-level expression occurred regardless of the pH of the culture medium. Analysis of the phosphatase spectrum by isoelectric focussing and enzyme activity staining both in the wild-type and thepacC disruptant showed that at least three acid phosphatases are regulated by thepacC. For the single alkaline phosphatase no pH regulation was observed.     

Inosine-5′-Monophosphate Dehydrogenase Is a Rate-determining Factor for p53-dependent Growth Regulation

We have proposed that reduced activity of inosine-5′-monophosphate dehydrogenase (IMPD; IMP:NAD oxidoreductase, EC 1.2.1.14), the rate-limiting enzyme for guanine nucleotide biosynthesis, in response to wild-type p53 expression, is essential for p53-dependent growth suppression. A gene transfer strategy was used to demonstrate that under physiological conditions constitutive IMPD expression prevents p53-dependent growth suppression. In these studies, expression of bax and waf1, genes implicated in p53-dependent growth suppression in response to DNA damage, remains elevated in response to p53. These findings indicate that under physiological conditions IMPD is a rate-determining factor for p53-dependent growth regulation. In addition, they suggest that the impd gene may be epistatic to bax and waf1 in growth suppression. Because of the role of IMPD in the production and balance of GTP and ATP, essential nucleotides for signal transduction, these results suggest that p53 controls cell division signals by regulating purine ribonucleotide metabolism.     

Ras oncogene enhances the production of a recombinant protein regulated by the cytomegalovirus promoter in BHK-21 cells

In order to enhance recombinant protein productivity in animal cells, we developed the oncogene activated production (OAP) system. The OAP system is based on the premise that oncogenes are able to enhance promoter activity. To this end, we constructed reported plasmids by fusing various promoters to the human interleukin-6 (hIL-6) cDNA, and the effector plasmids by inserting individual oncogenes, for example c-myc, c-fos, v-jun, v-myb and c-Ha-ras, downstream from the human cytomegalovirus immediate early (CMV) promoter. Results of transient expression experiments with BHK-21 cells suggest that the CMV promoter is the most potent promoter examined and that theras product is able to transactivate the -actin, CMV and SR promoters. Recombinant BHK-21 cells producing hIL-6 under the control of the CMV promoter were contransfected with theras oncogene and dihydrofolate reductase gene, then selected with 50 nM methotrexate to coamplify theras oncogene. We were able to rapidly establish a stable and highly productive clone which exhibited a 35-times higher production rate as compared to the control value.     

Isolation and sequence of a cDNA encoding mouse IMP dehydrogenase.

Inosinic acid (IMP) dehydrogenase (IMPD) catalyzes the conversion of IMP to XMP as the first committed step in GMP biosynthesis de novo. We have isolated a cDNA containing the complete coding region of mouse IMPD by its ability to complement a bacterial mutant lacking IMPD activity. Two independent cDNA clones were isolated by complementation, of which the longest was 1.7 kb in length. Northern analyses, using the IMPD cDNA as a probe, indicated that mature IMPD mRNA was a single species approx. 2.0 kb in size. Mouse IMPD is almost identical to Chinese hamster and human IMPDs and is highly conserved between Escherichia coli and mouse, with a direct amino acid (aa) identity of 39%, which increases to 60% if conserved aa are considered. The leader region of our longest cDNA clone is G + C-rich and contains two tandem copies of a G + C-rich direct repeat.     

Involvement of ras in sexual differentiation but not in growth control in fission yeast

The function of the ras⁺ gene of Schizosaccharomyces pombe has been studied by constructing null and activated alleles of this gene. An activated allele (^rasVal ₁₂) inhibits conjugation but has no effect on cell growth, entry into stationary phase or sporulation. The phenotype of ^rasVal ₁₂ is distinct from that caused by elevating the intracellular level of cAMP. This supports the hypothesis that ras of fission yeast does not modulate adenylate cyclase in a manner analogous to S. cerevisiae RAS. Introduction of a human ras sequence into fission yeast cells containing a non-functional null allele of ras restored the sexual differentiation process thus indicating that the human sequence can complement S. pombe ras. Our data suggest that although ras genes are highly conserved across a considerable evolutionary divide, the cellular function of the ras gene product varies in different organisms.     

A 610 kb YAC clone harbors 7 cM of tomato (Lycopersicon esculentum) DNA that includes themale sterile 14 gene and a hotspot for recombination

Pollen development requires both sporophytic and gametophytic gene expression. We are using a map-based cloning technique to isolate sporophytic genes which, when mutant, cause pollen abortion and a male sterile (ms) phenotype in tomato (Lycopersicon esculentum). We have genetically characterized onems locus (ms14) using RFLP analysis and identified flanking markers. High-resolution genomic physical mapping indicates that thems14 locus is located in a ～300 kb region. We have identified a YAC clone with an insert size of ～610 kb that contains thems14-linked markers, reflects the organization of the physical map and therefore most probably contains thems14 gene. In addition, we present evidence that the relationship between physical and genetic distance in this chromosomal region changes abruptly from ～105–140 kb/cM to less than 24 kb/cM, and suggest that the TG393-TG104 region is a hotspot for recombination.