Multiple determinants of eukaryotic mRNA stability

Regulated changes in mRNA stability play an important role in modulating the level of expression of many eukaryotic genes. In several systems, specific sequence determinants that dictate mRNA instability have been identified. Thus, the presence of instability determinants, and not the absence of sequences that dictate stability, appears to be required for regulated mRNA degradation. These instability determinants presumably interact with specific nucleases or other trans-acting factors that regulate the accessibility of the domain to nucleases. Although each RNA destabilization pathway has unique features, in many cases RNA degradation requires ongoing protein synthesis. In some of the systems discussed, the mRNAs are degraded co-translationally, perhaps by a ribosome-associated ribonuclease. For other messages, the mechanistic reasons for the dependence of mRNA degradation on ongoing protein synthesis are still unknown.     

TheAspergillus parasiticus polyketide synthase genepksA,a homolog ofAspergillus nidulans wA,is required for aflatoxin B1 biosynthesis

Aflatoxins comprise a group of polyketide-derived carcinogenic mycotoxins produced byAspergillus parasiticus andAspergillus flavus. By transformation with a disruption construct, pXX, we disrupted the aflatoxin pathway inA. parasiticus SRRC 2043, resulting in the inability of this strain to produce aflatoxin intermediates as well as a major yellow pigment in the transformants. The disruption was attributed to a single-crossover, homologous integration event between pXX and the recipientA. parasiticus genome at a specific locus, designatedpksA. Sequence analysis suggest thatpksA is a homolog of theAspergillus nidulans wA gene, a polyketide synthase gene involved in conidial wall pigment biosynthesis. The conservedβ-ketoacyl synthase, acyltransferase and acyl carrier-protein domains were present in the deduced amino acid sequence of thepksA product. Noβ-ketoacyl reductase and enoyl reductase domains were found, suggesting thatpksA does not encode catalytic activities for processingβ-carbon similar to those required for long chain fatty acid synthesis. ThepksA gene is located in the aflatoxin pathway gene cluster and is linked to thenor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin. These two genes are divergently transcribed from a 1.5 kb intergenic region. We propose thatpksA is a polyketide synthase gene required for the early steps of aflatoxin biosynthesis.     

Inactivation of erythropoietin receptor function by point mutations in a region having homology with other cytokine receptors.

The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a region, proximal to the transmembrane domain, that is essential for function and has homology with other members of the cytokine receptor family. To explore the functional significance of this region and to identify critical residues, we introduced several amino acid substitutions and examined their effects on erythropoietin-induced mitogenesis, tyrosine phosphorylation, and expression of immediate-early (c-fos, c-myc, and egr-1) and early (ornithine decarboxylase and T-cell receptor gamma) genes in interleukin-3-dependent cell lines. Amino acid substitution of W-282, which is strictly conserved at the middle portion of the homology region, completely abolished all the functions of the EpoR. Point mutation at L-306 or E-307, both of which are in a conserved LEVL motif, drastically impaired the function of the receptor in all assays. Other point mutations, introduced into less conserved amino acid residues, did not significantly impair the function of the receptor. These results demonstrate that conserved amino acid residues in this domain of the EpoR are required for mitogenesis, stimulation of tyrosine phosphorylation, and induction of immediate-early and early genes.     

Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus section Flavi.

The Aspergillus parasiticus aflR gene, a gene that may be involved in the regulation of aflatoxin biosynthesis, encodes a putative zinc finger DNA-binding protein. PCR and sequencing were used to examine the presence of aflR homologs in other members of Aspergillus Section Flavi. The predicted amino acid sequences indicated that the same zinc finger domain, CTSCASSKVRCTKEKPACARCIERGLAC, was present in all of the Aspergillus sojae, Aspergillus flavus, and Aspergillus parasiticus isolates examined and in some of the Aspergillus oryzae isolates examined. Unique base substitutions and a specific base deletion were found in the 5' untranslated and zinc finger region; these differences provided distinct fingerprints. A. oryzae and A. flavus had the T-G-A-A-X-C fingerprint, whereas A. parasiticus and A sojae had the C-C-C-C-C-T fingerprint at the corresponding positions. Specific nucleotides at positions -90 (C or T) and -132 (G or A) further distinguished A. flavus from A. oryzae and A. parasiticus from A. sojae, respectively. A sojae ATCC 9362, which was previously designated A. oryzae NRRL 1988, was determined to be a A. sojae strain on the basis of the presence of the characteristic fingerprint, A-C-C-C-C-C-C-T. The DNAs of other members of Aspergillus Section Flavi, such as Aspergillus nomius and Aspergillus tamarii, and some isolates of A. oryzae appeared to exhibit low levels of similarity to the A. parasiticus aflR gene since low amounts of PCR products or no PCR products were obtained when DNAs from these strains were used.     

Scanning electron microscopy of Treponema pallidum (Nichols strain) attached to cultured mammalian cells.

This paper describes the attachment of Treponema pallidum (Nichols strain) to cultured mammalian cells as a visualized by scanning electron microscopy. Treponemes were incubated for 3 hr with cultured cells derived from normal rabbit testes or human skin epithelium, then fixed, processed with critical-point drying, and examined with a Cambridge Mark 2A scanning electron microscope. Large numbers of treponemes became attached to the cultured cells without altering the morphological integrity of the cultured cells. Attachment appeared to involve a very close physical proximity of treponemes to the cultured cells; at the site of attachment, no changes such as swelling or indentation of the cultured cell surface were observed. The addition of ruthenium red to the fixatives produced a treponemal-associated surface precipitate. This material, which is probably mucopolysaccharide and/or phospholipid, may be important in protecting the organisms against host defense mechanisms; in addition, it may be involved in the serological unresponsiveness of freshly prepared suspensions of T. pallidum.     

Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes

A partial library of cloned human DNA was screened for sequences represented on and specific to the X chromosome. The library was constructed from Bam HI-digested human DNA from cells with X chromosome polyploidy, and was cloned in pBR322. The screening was performed by individually hybridizing ³²P-labeled cloned plasmids to Southern blots containing Bam HI-digested DNA from mouse-human hybrid cells having the human X chromosome and from derivative hybrids lacking the human X. Of 45 clones assayed, 33 contained sequences homologous to ones represented many times on the X. In situ hybridization to metaphase chromosomes demonstrated that at least four of these clones were homologous to autosomes as well. Only one of the 18 clones of this kind tested cross-hybridized with another. Two recombinant plasmids were shown to be derived from the X chromosome and to be X chromosome-specific by three criteria: they hybridized to a single band in the Southern blots of Bam HI-digested DNA from hybrid cells containing the X chromosome; they hybridized to a band of the same molecular weight as did the inserted DNA fragment; and they showed a dosage effect when hybridized to Southern blots of Bam HI-digested DNA from XY and XXX cells. One of these hybridized as a single-copy or low-order reiterated sequence in a Cot analysis using male DNA as driver. Our methods can be applied to the identification of any chromosome-specific clone. The two X-specific clones identified here should be useful in investigating the mechanism of X inactivation and in isolating a Barr body.