Regulation of cyclic peptide biosynthesis and pathogenicity in Cochliobolus carbonum by TOXEp, a novel protein with a bZIP basic DNA-binding motif and four ankyrin repeats

HC-toxin is an epoxide-containing cyclic tetrapeptide that is a critical virulence determinant in the pathogenic interaction between the filamentous fungus Cochliobolus carbonum and maize. HC-toxin exerts a potent cytostatic effect on plant and animal cells by inhibiting histone deacetylase. The biosynthesis of HC-toxin by C. carbonum is controlled by a complex genetic locus, TOX2, that contains multiple, duplicated copies of genes encoding export and biosynthetic enzymes. A new gene in the TOX2 complex, TOXE, has now been isolated. Mutation of TOXE by targeted gene disruption has no effect on growth and sporulation but abolishes HC-toxin production and pathogenicity. TOXE is required for the expression of three genes with a known or putative role in HC-toxin production, but is not required for expression of HTS1, which encodes the large, multifunctional peptide synthetase that is the central enzyme in HC-toxin biosynthesis. At its N-terminus, TOXEp has a bZIP basic DNA binding domain, but it does not contain any discernible leucine zipper or helix-loop-helix. At its carboxy terminus, TOXEp contains four ankyrin repeats. In having these two common regulatory motifs in a single polypeptide, TOXEp appears to represent a novel class of regulatory protein. TOXE is present only in HC-toxin-producing (Tox2⁺) isolates of C. carbonum. Most Tox2⁺ isolates have two copies; in strain SB111, one copy of TOXE is on the same 3.5-Mb chromosome that contains all of the other genes known to be involved in HC-toxin biosynthesis, and the second copy of TOXE is on a 0.7-Mb chromosome.     

Regulation of cyclic peptide biosynthesis and pathogenicity in Cochliobolus carbonum by TOXEp, a novel protein with a bZIP basic DNA-binding motif and four ankyrin repeats

HC-toxin is an epoxide-containing cyclic tetrapeptide that is a critical virulence determinant in the pathogenic interaction between the filamentous fungus Cochliobolus carbonum and maize. HC-toxin exerts a potent cytostatic effect on plant and animal cells by inhibiting histone deacetylase. The biosynthesis of HC-toxin by C. carbonum is controlled by a complex genetic locus, TOX2, that contains multiple, duplicated copies of genes encoding export and biosynthetic enzymes. A new gene in the TOX2 complex, TOXE, has now been isolated. Mutation of TOXE by targeted gene disruption has no effect on growth and sporulation but abolishes HC-toxin production and pathogenicity. TOXE is required for the expression of three genes with a known or putative role in HC-toxin production, but is not required for expression of HTS1, which encodes the large, multifunctional peptide synthetase that is the central enzyme in HC-toxin biosynthesis. At its N-terminus, TOXEp has a bZIP basic DNA binding domain, but it does not contain any discernible leucine zipper or helix-loop-helix. At its carboxy terminus, TOXEp contains four ankyrin repeats. In having these two common regulatory motifs in a single polypeptide, TOXEp appears to represent a novel class of regulatory protein. TOXE is present only in HC-toxin-producing (Tox2⁺) isolates of C. carbonum. Most Tox2⁺ isolates have two copies; in strain SB111, one copy of TOXE is on the same 3.5-Mb chromosome that contains all of the other genes known to be involved in HC-toxin biosynthesis, and the second copy of TOXE is on a 0.7-Mb chromosome. Received: 20 April 1998 / Accepted: 21 September 1998     

MZF6D, a novel KRAB zinc-finger gene expressed exclusively in meiotic male germ cells

Spermatogenesis takes place in the seminiferous tubule in the testes and culminates in the production of spermatozoa (male gametes). Here we report the identification of a novel mouse zinc-finger gene, MZF6D, which is selectively expressed in meiotic spermatocytes. The MZF6D protein contains an N-terminally located repressor domain, a KRAB domain, followed by at least seven successive Krüppel zinc-finger motifs. The KRAB domain of MZF6D, which consists of a KRAB A box and the newly identified KRAB C box, has previously been shown to interact with TIF1beta, which is the common corepressor of all KRAB zinc-finger proteins. Northern blot analysis shows that the expression of MZF6D is restricted to testes. This was confirmed by RT-PCR analysis of a panel of mouse tissues. In situ hybridization of sections from adult mouse testes localizes the expression to meiotic spermatocytes, suggesting a specific role for MZF6D in the regulation of spermatogenesis.     

LETM1, a gene deleted in Wolf-Hirschhorn syndrome, encodes an evolutionarily conserved mitochondrial protein

The leucine zipper-, EF-hand-containing transmembrane protein 1 (LETM1) has recently been cloned in an attempt to identify genes deleted in Wolf-Hirschhorn syndrome (WHS), a microdeletion syndrome characterized by severe growth and mental retardation, hypotonia, seizures, and typical facial dysmorphic features. LETM1 is deleted in almost all patients with the full phenotype and has recently been suggested as an excellent candidate gene for the seizures in WHS patients. We have shown that LETM1 is evolutionarily conserved throughout the eukaryotic kingdom and exhibits homology to MDM38, a putative yeast protein involved in mitochondrial morphology. Using LETM1-EGFP fusion constructs and an anti-rat LetM1 polyclonal antibody we have demonstrated that LETM1 is located in the mitochondria. The present study presents information about a possible function for LETM1 and suggests that at least some (neuromuscular) features of WHS may be caused by mitochondrial dysfunction.     

cDNA cloning and expression of oxysterol-binding protein, an oligomer with a potential leucine zipper

Feedback repression of the genes encoding the low density lipoprotein receptor and several enzymes of the cholesterol biosynthetic pathway is mediated by 25-hydroxycholesterol and other oxysterols. In this study, we have cloned a rabbit cDNA encoding an oxysterol-binding protein that may play a role in this regulation. The predicted amino acid sequence revealed a protein of 809 amino acids with two distinctive features: 1) a glycine- and alanine-rich region (63% of 80 residues) at the NH2 terminus, and 2) a 35-residue leucine zipper motif that may mediate the previously observed oligomerization of the protein. When transfected into simian COS cells, the rabbit cDNA produced a protein that exhibited the same affinity and specificity for sterols as the previously purified hamster liver protein. Immunoblotting analysis showed that the rabbit cDNA encodes both the 96- and 101-kilodalton forms of the oxysterol-binding protein that were previously observed. The availability of an expressible cDNA for the oxysterol-binding protein should help elucidate its role in sterol metabolism.     

Molecular characterization of HymA, an evolutionarily highly conserved and highly expressed protein of Aspergillus nidulans

Aspergillus nidulans reproduces asexually via uninucleate, haploid spores, which are produced on morphologically differentiated aerial structures, called conidiophores. These consist of four distinct cell types, a foot with a terminally swollen stalk, metulae, phialides and conidiospores. The molecular mechanisms underlying the morphological changes that occur during conidiophore development have been studied by mutant analysis. We have isolated the hymA mutant, in which conidiophore development is affected at the metula stage. In the mutant metulae do not differentiate properly but come to resemble hyphae (hym?=?hypha-like metulae). In this paper we have analyzed the corresponding gene. It encodes a highly expressed 44?kDa protein which resides in the cytoplasm and has homologues in yeast, plants, fly, worm, fish, mice and man. We constructed hym deletion strains of Saccharomyces cerevisiae and of A. nidulans and found that the gene is essential in S. cerevisiae but is dispensable in the filamentous fungus. A cellular function for the Hym protein has not yet been defined in any organism. To demonstrate functional conservation we constructed a chimeric protein comprised of the N-terminal half of the A.?nidulans and the C-terminal half of the mouse homologue MO25. This hybrid protein could fully substitute for HymA function in A. nidulans. In addition, the mouse protein itself partially rescued the hymA mutation in the fungus. HymA is thus highly conserved in evolution and probably serves similar functions. The fact that hymA is required for conidiophore development in A. nidulans suggests that homologous genes in other organisms might also be involved in morphogenesis.     

Characterization and expression of the gene encoding membralin, an evolutionary conserved protein expressed in the central nervous system

We have identified a novel gene that encodes an evolutionary conserved protein that we name membralin since it contains multiple transmembrane regions. The human gene C19orf6 localizes to chromosome 19p13.3. Splice variant membralin-1 is encoded by 11 exons, translating into 620 amino acids. In addition, we found evidence for two additional splice variants in the human. The mouse gene ORF61 localizes to chromosome 10. We cloned two splice variants in mouse: membralin-1, which is encoded by 12 exons, translating into 574 amino acids, and membralin-2, which translates into 598 amino acids. The existence of rat membralin-1 (574 amino acids long) is, so far, only supported by in situ hybridization result, whereas the existence of rat membralin-2 (598 amino acids long) is strongly supported by overlapping ESTs. Gene homologues were also identified in fruit-fly (CG8405, chromosome 2R 52; two splice variants), nematode (chromosome III), and Arabidopsis thaliana (chromosome 1). Sequence analysis revealed no closely related genes, suggesting that membralin represents the sole member of a unique protein family.     

Epstein-Barr virus infection induces expression in B lymphocytes of a novel gene encoding an evolutionarily conserved 55-kilodalton actin-bundling protein.

A novel human mRNA whose expression is induced over 200-fold in B lymphocytes by latent Epstein-Barr virus (EBV) infection was reverse transcribed, cloned, and sequenced. The mRNA is predicted to encode a protein containing four peptides which precisely match amino acid sequences from a previously identified 55-kDa actin-bundling protein, p55. In vitro translation of the cDNA results in a 55-kDa protein which binds to actin filaments in the presence of purified p55 from HeLa cells. The p55 mRNA is undetectable in non-EBV-infected B- and T-cell lines or in a myelomonocytic cell line (U937). Newly infected primary human B lymphocytes, EBV-transformed B-cell lines, latently infected Burkitt tumor cells expressing EBNA2 and LMP1, a chronic myelogenous leukemia cell line (K562), and an osteosarcoma cell line (TK143) contain high levels of p55 mRNA or protein. In EBV-transformed B cells, p55 localizes to perinuclear cytoplasm and to cell surface processes that resemble filopodia. The p55 mRNA is detected at high levels in spleen and brain tissues, at moderate levels in lung and placenta tissues, and at low levels in skeletal muscle, liver, and tonsil tissues and is undetectable in heart, kidney, pancreas, and bone marrow tissues. Immunohistochemical staining of human brain tissue demonstrates p55 localization to the perinuclear cytoplasm and dendritic processes of many, but not all, types of cortical or cerebellar neurons, to glial cells, and to capillary endothelial cells. In cultured primary rat neurons, p55 is distributed throughout the perinuclear cytoplasm and in subcortical filamentous structures of dendrites and growth cones. p55 is highly evolutionarily conserved since it shows 40% amino acid sequence identity to the Drosophila singed gene product and 37% identity to fascin, an echinoderm actin-bundling protein. The evolutionary conservation of p55 and its lack of extensive homology to other actin-binding proteins suggest that p55 has specific microfilament-associated functions in cells in which it is differentially expressed, including neural cells and EBV-transformed B lymphocytes.     

Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells

The gene expression patterns of primordial germ cells (PGCs) and embryonic stem cells were analyzed by a modified serial analysis of gene expression. During the process, we cloned a novel gene, PGC7, which was preferentially expressed in PGCs. Immunohistochemical analysis revealed that PGC7 was specifically expressed in early pre-implantation embryos, PGCs and oocytes. These results suggest that PGC7 might play an important role in the development of PGCs and oocytes.     

Poplar metal tolerance protein 1 confers zinc tolerance and is an oligomeric vacuolar zinc transporter with an essential leucine zipper motif

Cation diffusion facilitator (CDF) proteins are a recently discovered family of cation efflux transporters that might play an essential role in metal homeostasis and tolerance. Here, we describe the identification, characterization, and localization of PtdMTP1, a member of the CDF family from the hybrid poplar Populus trichocarpa x Populus deltoides. PtdMTP1 is expressed constitutively and ubiquitously, although at low levels. Heterologous expression in yeast showed that PtdMTP1 was able to complement the hypersensitivity of mutant strains to Zn but not to other metals, including Cd, Co, Mn, and Ni. PtdMTP1 fused to green fluorescent protein localized to the vacuolar membrane both in yeast and in plant cells, consistent with a function of PtdMTP1 in zinc sequestration. Overexpression of PtdMTP1 in Arabidopsis confers Zn tolerance. We show that PtdMTP1, when expressed in yeast and Arabidopsis, forms homooligomers, a novel feature of CDF members. Oligomer formation is disrupted by reducing agents, indicating possible disulfide bridge formation. PtdMTP1 also contains a conserved Leu zipper motif. Although not necessary for oligomer formation, Leu residues within this motif are required for PtdMTP1 functional activity.