Structures of two molluscan hemocyanin genes: Significance for gene evolution

We present here the description of genes coding for molluscan hemocyanins. Two distantly related mollusks, Haliotis tuberculata and Octopus dofleini, were studied. The typical architecture of a molluscan hemocyanin subunit, which is a string of seven or eight globular functional units (FUs, designated a to h, about 50 kDa each), is reflected by the gene organization: a series of eight structurally related coding regions in Haliotis, corresponding to FU-a to FU-h, with seven highly variable linker introns of 174 to 3,198 bp length (all in phase 1). In Octopus seven coding regions (FU-a to FU-g) are found, separated by phase 1 introns varying in length from 100 bp to 910 bp. Both genes exhibit typical signal (export) sequences, and in both cases these are interrupted by an additional intron. Each gene also contains an intron between signal peptide and FU-a and in the 3′ untranslated region. Of special relevance for evolutionary considerations are introns interrupting those regions that encode a discrete functional unit. We found that five of the eight FUs in Haliotis each are encoded by a single exon, whereas FU-f, FU-g, and FU-a are encoded by two, three and four exons, respectively. Similarly, in Octopus four of the FUs each correspond to an uninterrupted exon, whereas FU-b, FU-e, and FU-f each contain a single intron. Although the positioning of the introns between FUs is highly conserved in the two mollusks, the introns within FUs show no relationship either in location nor phase. It is proposed that the introns between FUs were generated as the eight-unit polypeptide evolved from a monomeric precursor, and that the internal introns have been added later. A hypothesis for evolution of the ring-like quaternary structure of molluscan hemocyanins is presented.

Long-Day Up-Regulation of a GAMYB Gene during Lolium temulentum Inflorescence Formation

Long-day exposure of the grass Lolium temulentum may regulate flowering via changes in gibberellin (GA) levels. Therefore, we have examined both GA levels and expression of a MYB transcription factor that is specific to the GA signal transduction pathway in monocots. This MYB gene from L. temulentum shows over 90% nucleotide identity with the barley and rice GAMYB genes, and, like them, gibberellic acid (GA3) up-regulates its expression in the seed. Furthermore, cDNAs of both the barley and L. temulentum GAMYB show the same simple patterns of hybridization with digests of L. temulentum genomic DNA. Compared with vegetative shoot apices of L. temulentum, the in situ mRNA expression of LtGAMYB does not change during the earliest steps of “floral” initiation at the apex. However, by 100 h (the double-ridge stage of flowering) its expression increased substantially and was highest in the terminal and lateral spikelet sites. Thereafter, expression declined overall but then increased within stamen primordia. Prior to increased LtGAMYB expression, long-day exposure sufficient to induce flowering led to increased (5- to 20-fold) levels of GA1 and GA4 in the leaf. Thus, increases first in GA level in the leaf followed by increased expression of LtGAMYB in the apex suggest important signaling and/or response roles in flowering.

Overexpression of a Novel Arabidopsis Gene Related to Putative Zinc-Transporter Genes from Animals Can Lead to Enhanced Zinc Resistance and Accumulation

We describe the isolation of an Arabidopsis gene that is closely related to the animal ZnT genes (Zn transporter). The protein encoded by the ZAT (Zn transporter of Arabidopsis thaliana) gene has 398 amino acid residues and is predicted to have six membrane-spanning domains. To obtain evidence for the postulated function of the Arabidopsis gene, transgenic plants with the ZAT coding sequence under control of the cauliflower mosaic virus 35S promoter were analyzed. Plants obtained with ZAT in the sense orientation exhibited enhanced Zn resistance and strongly increased Zn content in the roots under high Zn exposure. Antisense mRNA-producing plants were viable, with a wild-type level of Zn resistance and content, like plants expressing a truncated coding sequence lacking the C-terminal cytoplasmic domain of the protein. The availability of ZAT can lead to a better understanding of the mechanism of Zn homeostasis and resistance in plants.

The Arabidopsis CBF Gene Family Is Composed of Three Genes Encoding AP2 Domain-Containing Proteins Whose Expression Is Regulated by Low Temperature but Not by Abscisic Acid or Dehydration1

We have identified two genes from Arabidopsis that show high similarity with CBF1, a gene encoding an AP2 domain-containing transcriptional activator that binds to the low-temperature-responsive element CCGAC and induces the expression of some cold-regulated genes, increasing plant freezing tolerance. These two genes, which we have named CBF2 and CBF3, also encode proteins containing AP2 DNA-binding motifs. Furthermore, like CBF1, CBF2 and CBF3 proteins also include putative nuclear-localization signals and potential acidic activation domains. The CBF2 and CBF3 genes are linked to CBF1, constituting a cluster on the bottom arm of chromosome IV. The high level of similarity among the three CBF genes, their tandem organization, and the fact that they have the same transcriptional orientation all suggest a common origin. CBF1, CBF2, and CBF3 show identical expression patterns, being induced very rapidly by low-temperature treatment. However, in contrast to most of the cold-induced plant genes characterized, they are not responsive to abscisic acid or dehydration. Taken together, all of these data suggest that CBF2 and CBF3 may function as transcriptional activators, controlling the level of low-temperature gene expression and promoting freezing tolerance through an abscisic acid-independent pathway.

Ectopic Expression of the Maize Homeobox Gene Liguleless3 Alters Cell Fates in the Leaf1

The semidominant mutation Liguleless3-O (Lg3-O) causes a blade-to-sheath transformation at the midrib region of the maize (Zea mays L.) leaf. We isolated a full-length lg3 cDNA containing a knotted1-like family homeobox. Six Lg3-O partial revertant alleles caused by insertion of a Mutator (Mu) transposon and two deletion derivatives were isolated and used to verify that our knotted1-like cDNA corresponds to the LG3 message. In wild-type plants the LG3 mRNA is expressed in apical regions but is not expressed in leaves. In mutant plants harboring any of three dominant lg3 alleles (Lg3-O, -Mlg, and -347), LG3 mRNA is expressed in leaf sheath tissue, indicating that the Lg3 phenotype is due to ectopic expression of the gene. The Lg3-O revertant alleles represent two classes of Lg3 phenotypes that correlate well with the level of ectopic Lg3 expression. High levels of ectopic LG3 mRNA expression results in a severe Lg3 phenotype, whereas weak ectopic Lg3 expression results in a mild Lg3 phenotype. We propose that ectopic Lg3 expression early in leaf development causes the blade-to-sheath transformation, but the level of expression determines the extent of the transformation.

The Down-Regulation of Mt4-Like Genes by Phosphate Fertilization Occurs Systemically and Involves Phosphate Translocation to the Shoots1

Mt4 is a cDNA representing a phosphate-starvation-inducible gene from Medicago truncatula that is down-regulated in roots in response to inorganic phosphate (Pi) fertilization and colonization by arbuscular mycorrhizal fungi. Split-root experiments revealed that the expression of the Mt4 gene in M. truncatula roots is down-regulated systemically by both Pi fertilization and colonization by arbuscular mycorrhizal fungi. A comparison of Pi levels in these tissues suggested that this systemic down-regulation is not caused by Pi accumulation. Using a 30-bp region of the Mt4 gene as a probe, Pi-starvation-inducible Mt4-like genes were detected in Arabidopsis and soybean (Glycine max L.), but not in corn (Zea mays L.). Analysis of the expression of the Mt4-like Arabidopsis gene, At4, in wild-type Arabidopsis and pho1, a mutant unable to load Pi into the xylem, suggests that Pi must first be translocated to the shoot for down-regulation to occur. The data from the pho1 and split-root studies are consistent with the presence of a translocatable shoot factor responsible for mediating the systemic down-regulation of Mt4-like genes in roots.

The Schizosaccharomyces pombe spo20+ Gene Encoding a Homologue of Saccharomyces cerevisiae Sec14 Plays an Important Role in Forespore Membrane Formation

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20+ is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20+ gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

Ubc9 interacts with a nuclear localization signal and mediates nuclear localization of the paired-like homeobox protein Vsx-1 independent of SUMO-1 modification

Vsx-1 is a paired-like:CVC homeobox gene whose expression is linked to bipolar cell differentiation during zebrafish retinogenesis. We used a yeast two-hybrid screen to identify proteins interacting with Vsx-1 and isolated Ubc9, an enzyme that conjugates the small ubiquitin-like modifier SUMO-1. Despite its interaction with Ubc9, we show that Vsx-1 is not a substrate for SUMO-1 in COS-7 cells or in vitro. When a yeast two-hybrid assay is used, deletion analysis of the interacting domain on Vsx-1 shows that Ubc9 binds to a nuclear localization signal (NLS) at the NH2 terminus of the homeodomain. In SW13 cells, Vsx-1 localizes to the nucleus and is excluded from nucleoli. Deletion of the NLS disrupts this nuclear localization, resulting in a diffuse cytoplasmic distribution of Vsx-1. In SW13 AK1 cells that express low levels of endogenous Ubc9, Vsx-1 accumulates in a perinuclear ring and colocalizes with an endoplasmic reticulum marker. However, NLS-tagged STAT1 protein exhibits normal nuclear localization in both SW13 and SW13 AK1 cells, suggesting that nuclear import is not globally disrupted. Cotransfection of Vsx-1 with Ubc9 restores Vsx-1 nuclear localization in SW3 AK1 cells and demonstrates that Ubc9 is required for the nuclear localization of Vsx-1. Ubc9 continues to restore nuclear localization even after a C93S active site mutation has eliminated its SUMO-1-conjugating ability. These results suggest that Ubc9 mediates the nuclear localization of Vsx-1, and possibly other proteins, through a nonenzymatic mechanism that is independent of SUMO-1 conjugation.

Considerations of transcriptional control mechanisms: Do TFIID–core promoter complexes recapitulate nucleosome-like functions?

The general transcription initiation factor TFIID was originally identified, purified, and characterized with a biochemical assay in which accurate transcription initiation is reconstituted with multiple, chromatographically separable activities. Biochemical analyses have demonstrated that TFIID is a multiprotein complex that directs preinitiation complex assembly on both TATA box-containing and TATA-less promoters, and some TFIID subunits have been shown to be molecular targets for activation domains in DNA-binding regulatory proteins. These findings have most commonly been interpreted to support the view that transcriptional activation by upstream factors is the result of enhanced TFIID recruitment to the core promoter. Recent insights into the architecture and cell-cycle regulation of the multiprotein TFIID complex prompt both a reassessment of the functional role of TFIID in gene activation and a review of some of the less well-appreciated literature on TFIID. We present a speculative model for diverse functional roles of TFIID in the cell, explore the merits of the model in the context of published data, and suggest experimental approaches to resolve unanswered questions. Finally, we point out how the proposed functional roles of TFIID in eukaryotic class II transcription fit into a model for promoter recognition and activation that applies to both eubacteria and eukaryotes.

Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode α1,4- N-acetylglucosaminyltransferases that likely are involved in heparan sulfate/ heparin biosynthesis

The tumor suppressors EXT1 and EXT2 are associated with hereditary multiple exostoses and encode bifunctional glycosyltransferases essential for chain polymerization of heparan sulfate (HS) and its analog, heparin (Hep). Three highly homologous EXT-like genes, EXTL1–EXTL3, have been cloned, and EXTL2 is an α1,4-GlcNAc transferase I, the key enzyme that initiates the HS/Hep synthesis. In the present study, truncated forms of EXTL1 and EXTL3, lacking the putative NH2-terminal transmembrane and cytoplasmic domains, were transiently expressed in COS-1 cells and found to harbor α-GlcNAc transferase activity. EXTL3 used not only N-acetylheparosan oligosaccharides that represent growing HS chains but also GlcAβ1–3Galβ1-O-C2H4NH-benzyloxycarbonyl (Cbz), a synthetic substrate for α-GlcNAc transferase I that determines and initiates HS/Hep synthesis. In contrast, EXTL1 used only the former acceptor. Neither EXTL1 nor EXTL3 showed any glucuronyltransferase activity as examined with N-acetylheparosan oligosaccharides. Heparitinase I digestion of each transferase-reaction product showed that GlcNAc had been transferred exclusively through an α1,4-configuration. Hence, EXTL3 most likely is involved in both chain initiation and elongation, whereas EXTL1 possibly is involved only in the chain elongation of HS and, maybe, Hep as well. Thus, their acceptor specificities of the five family members are overlapping but distinct from each other, except for EXT1 and EXT2 with the same specificity. It now has been clarified that all of the five cloned human EXT gene family proteins harbor glycosyltransferase activities, which probably contribute to the synthesis of HS and Hep.

A Nitrilase-Like Protein Interacts with GCC Box DNA-Binding Proteins Involved in Ethylene and Defense Responses1

Ethylene-responsive element-binding proteins (EREBPs) of tobacco (Nicotiana tabacum L.) bind to the GCC box of many pathogenesis-related (PR) gene promoters, including osmotin (PR-5). The two GCC boxes on the osmotin promoter are known to be required, but not sufficient, for maximal ethylene responsiveness. EREBPs participate in the signal transduction pathway leading from exogenous ethylene application and pathogen infection to PR gene induction. In this study EREBP3 was used as bait in a yeast two-hybrid interaction trap with a tobacco cDNA library as prey to isolate signal transduction pathway intermediates that interact with EREBPs. One of the strongest interactors was found to encode a nitrilase-like protein (NLP). Nitrilase is an enzyme involved in auxin biosynthesis. NLP interacted with other EREBP family members, namely tobacco EREBP2 and tomato (Lycopersicon esculentum L.) Pti4/5/6. The EREBP2-EREBP3 interaction with NLP required part of the DNA-binding domain. The specificity of interaction was further confirmed by protein-binding studies in solution. We propose that the EREBP-NLP interaction serves to regulate PR gene expression by sequestration of EREBPs in the cytoplasm.

(+)-Abscisic Acid Metabolism, 3-Ketoacyl-Coenzyme A Synthase Gene Expression, and Very-Long-Chain Monounsaturated Fatty Acid Biosynthesis in Brassica napus Embryos1

Microspore-derived embryos of Brassica napus cv Reston were used to examine the effects of exogenous (+)-abscisic acid (ABA) and related compounds on the accumulation of very-long-chain monounsaturated fatty acids (VLCMFAs), VLCMFA elongase complex activity, and induction of the 3-ketoacyl-coenzyme A synthase (KCS) gene encoding the condensing enzyme of the VLCMFA elongation system. Of the concentrations tested, (+)-ABA at 10 μm showed the strongest effect. Maximum activity of the elongase complex, observed 6 h after 10 μm (+)-ABA treatment, was 60% higher than that of the untreated embryos at 24 h. The transcript of the KCS gene was induced by 10 μm (+)-ABA within 1 h and further increased up to 6 h. The VLCMFAs eicosenoic acid (20:1) and erucoic acid (22:1) increased by 1.5- to 2-fold in embryos treated with (+)-ABA for 72 h. Also, (+)-8′-methylene ABA, which is metabolized more slowly than ABA, had a stronger ABA-like effect on the KCS gene transcription, elongase complex activity (28% higher), and level of VLCMFAs (25–30% higher) than ABA. After 24 h approximately 60% of the added (+)-[3H]ABA (10 μm) was metabolized, yielding labeled phaseic and dihydrophaseic acid. This study demonstrates that (+)-ABA promotes VLCMFA biosynthesis via increased expression of the KCS gene and that reducing ABA catabolism would increase VLCMFAs in microspore-derived embryos.

Expression of an Arabidopsis Aspartate Kinase/Homoserine Dehydrogenase Gene Is Metabolically Regulated by Photosynthesis-Related Signals but Not by Nitrogenous Compounds1

Although the control of carbon fixation and nitrogen assimilation has been studied in detail, relatively little is known about the regulation of carbon and nitrogen flow into amino acids. In this paper we report our study of the metabolic regulation of expression of an Arabidopsis aspartate kinase/homoserine dehydrogenase (AK/HSD) gene, which encodes two linked key enzymes in the biosynthetic pathway of aspartate family amino acids. Northern blot analyses, as well as expression of chimeric AK/HSD-β-glucuronidase constructs, have shown that the expression of this gene is regulated by the photosynthesis-related metabolites sucrose and phosphate but not by nitrogenous compounds. In addition, analysis of AK/HSD promoter deletions suggested that a CTTGACTCTA sequence, resembling the binding site for the yeast GCN4 transcription factor, is likely to play a functional role in the expression of this gene. Nevertheless, longer promoter fragments, lacking the GCN4-like element, were still able to confer sugar inducibility, implying that the metabolic regulation of this gene is apparently obtained by multiple and redundant promoter sequences. The present and previous studies suggest that the conversion of aspartate into either the storage amino acid asparagine or aspartate family amino acids is subject to a coordinated, reciprocal metabolic control, and this biochemical branch point is a part of a larger, coordinated regulatory mechanism of nitrogen and carbon storage and utilization.

Studying Early Nodulin Gene ENOD40 Expression and Induction by Nodulation Factor and Cytokinin in Transgenic Alfalfa1

ENOD40, an early nodulin gene, is expressed following inoculation with Rhizobium meliloti or by adding R. meliloti-produced nodulation (Nod) factors or the plant hormone cytokinin to uninoculated roots. We isolated two MsENOD40 clones, designated MsENOD40–1 and MsENOD40–2, with distinct promoters from an alfalfa (Medicago sativa cv Chief) genomic library. The promoters were fused to the reporter gene uidA (gus), and the constructs were introduced into alfalfa. We observed that the MsENOD40–1 construct was expressed almost exclusively under symbiotic conditions. The MsENOD40–2 construct was transcribed under both symbiotic and nonsymbiotic conditions and in nonnodular and nodular tissues. Both MsENOD40 promoter-gus constructs were similarly expressed as nodules developed, and both were expressed in roots treated with 6-benzylaminopurine or purified Nod factor. However, no blue color was detected in nodule-like structures induced by the auxin transport inhibitor N-1-(naphthyl)phthalamic acid on roots of plants containing the MsENOD40–1 promoter construct, whereas pseudonodules from plants containing the MsENOD40–2 promoter construct stained blue. A 616-bp region at the distal 5′ end of the promoter is important for proper spatial expression of MsENOD40 in nodules and also for Nod-factor and cytokinin-induced expression.

A yeast gene, MGS1, encoding a DNA-dependent AAA+ ATPase is required to maintain genome stability

Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases. If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability. Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans. Here we report a Saccharomyces cerevisiae gene, MGS1 (Maintenance of Genome Stability 1), which encodes a protein belonging to the AAA+ class of ATPases, and whose central region is similar to Escherichia coli RuvB, a Holliday junction branch migration motor protein. The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes. The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities. An mgs1 deletion mutant has an elevated rate of mitotic recombination, which causes genome instability. The mgs1 mutation is synergistic with a mutation in top3 (encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability. In contrast to the mgs1 mutation, a mutation in the sgs1 gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the top3 mutant. Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability.