Leaf-specifically expressed genes for polypeptides destined for chloroplasts with domains of σ70 factors of bacterial RNA polymerases in Arabidopsis thaliana

Genes for σ-like factors of bacterial-type RNA polymerase have not been characterized from any multicellular eukaryotes, although they probably play a crucial role in the expression of plastid photosynthesis genes. We have cloned three distinct cDNAs, designated SIG1, SIG2, and SIG3, for polypeptides possessing amino acid sequences for domains conserved in σ70 factors of bacterial RNA polymerases from the higher plant Arabidopsis thaliana. Each gene is present as one copy per haploid genome without any additional sequences hybridized in the genome. Transient expression assays using green fluorescent protein demonstrated that N-terminal regions of the SIG2 and SIG3 ORFs could function as transit peptides for import into chloroplasts. Transcripts for all three SIG genes were detected in leaves but not in roots, and were induced in leaves of dark-adapted plants in rapid response to light illumination. Together with results of our previous analysis of tissue-specific regulation of transcription of plastid photosynthesis genes, these results indicate that expressed levels of the genes may influence transcription by regulating RNA polymerase activity in a green tissue-specific manner.

Phototactic Migration of Dictyostelium Cells Is Linked to a New Type of Gelsolin-related Protein

The molecular and functional characterization of a 125-kDa Ca2+-extractable protein of the Triton X-100–insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type in Dictyostelium, as well as in heterologous cells from lower to higher eukaryotes.

MCD4 Encodes a Conserved Endoplasmic Reticulum Membrane Protein Essential for Glycosylphosphatidylinositol Anchor Synthesis in Yeast

Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p’s lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4–174) harbors a single amino acid change in motif 2. The mcd4–174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4–174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4–174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.

Short DNA Fragments without Sequence Similarity Are Initiation Sites for Replication in the Chromosome of the Yeast Yarrowia lipolytica

We have previously shown that both a centromere (CEN) and a replication origin are necessary for plasmid maintenance in the yeast Yarrowia lipolytica (Vernis et al., 1997). Because of this requirement, only a small number of centromere-proximal replication origins have been isolated from Yarrowia. We used a CEN-based plasmid to obtain noncentromeric origins, and several new fragments, some unique and some repetitive sequences, were isolated. Some of them were analyzed by two-dimensional gel electrophoresis and correspond to actual sites of initiation (ORI) on the chromosome. We observed that a 125-bp fragment is sufficient for a functional ORI on plasmid, and that chromosomal origins moved to ectopic sites on the chromosome continue to act as initiation sites. These Yarrowia origins share an 8-bp motif, which is not essential for origin function on plasmids. The Yarrowia origins do not display any obvious common structural features, like bent DNA or DNA unwinding elements, generally present at or near eukaryotic replication origins. Y. lipolytica origins thus share features of those in the unicellular Saccharomyces cerevisiae and in multicellular eukaryotes: they are discrete and short genetic elements without sequence similarity.

Trypanosoma cruzi Calreticulin Is a Lectin That Binds Monoglucosylated Oligosaccharides but Not Protein Moieties of Glycoproteins

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse–chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin–cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-β-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin–cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.

Gene Conversion of Major Histocompatibility Complex Genes in the Mouse Spermatogenesis is a Premeiotic Event

The molecular genetic mechanism of gene conversion in higher eukaryotes remains unknown. We find it of considerable interest to determine when during spermatogenesis gene conversion occurs. We have therefore purified pachytene spermatocytes and haploid spermatocytes from adult mice and analyzed these fractions for the presence of gene conversion products resulting from the transfer between the major histocompatibility complex class II genes Ebd and Abk in a polymerase chain reaction assay. We have further isolated spermatogenic cells from prepubescent mice and analyzed them for the presence of the same gene conversion products. We can detect gene conversion products in testis cells as early as in 8-d-old mice where the only existing spermatogenic cells are spermatogonia. The frequency of gene conversion products remains the same as the cells reach meiosis in 18-d-old mice, and is unchanged after meiosis is completed in haploid spermatocytes. Gene conversion of this specific fragment therefore appears to be a premeiotic event and, consequently, relies on genetic mechanisms other than normal meiotic recombination.

Characterization of Saccharomyces cerevisiae dna2 Mutants Suggests a Role for the Helicase Late in S Phase

The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like “lipid kinase” domain and are required for early G1 progression in eukaryotes. Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p). This gene is identical to DNA2, encoding a helicase required for DNA replication. As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants. Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions. Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and MEC1-dependent manner, suggesting that Dna2p has a role in S phase. Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis. Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2 mutants are not deficient in bulk DNA synthesis. These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation.

An ancestral MADS-box gene duplication occurred before the divergence of plants and animals

Changes in genes encoding transcriptional regulators can alter development and are important components of the molecular mechanisms of morphological evolution. MADS-box genes encode transcriptional regulators of diverse and important biological functions. In plants, MADS-box genes regulate flower, fruit, leaf, and root development. Recent sequencing efforts in Arabidopsis have allowed a nearly complete sampling of the MADS-box gene family from a single plant, something that was lacking in previous phylogenetic studies. To test the long-suspected parallel between the evolution of the MADS-box gene family and the evolution of plant form, a polarized gene phylogeny is necessary. Here we suggest that a gene duplication ancestral to the divergence of plants and animals gave rise to two main lineages of MADS-box genes: TypeI and TypeII. We locate the root of the eukaryotic MADS-box gene family between these two lineages. A novel monophyletic group of plant MADS domains (AGL34 like) seems to be more closely related to previously identified animal SRF-like MADS domains to form TypeI lineage. Most other plant sequences form a clear monophyletic group with animal MEF2-like domains to form TypeII lineage. Only plant TypeII members have a K domain that is downstream of the MADS domain in most plant members previously identified. This suggests that the K domain evolved after the duplication that gave rise to the two lineages. Finally, a group of intermediate plant sequences could be the result of recombination events. These analyses may guide the search for MADS-box sequences in basal eukaryotes and the phylogenetic placement of new genes from other plant species.

A possible mitochondrial gene in the early-branching amitochondriate protist Trichomonas vaginalis

Trichomonads are anaerobic flagellated protists that, based on analyses of ribosomal RNA sequences, represent one of the earliest branching lineages among the eukaryotes. The absence of mitochondria in these organisms coupled with their deep phylogenetic position has prompted several authors to suggest that trichomonads, along with other deeply-branching amitochondriate protist groups, diverged from the main eukaryotic lineage prior to the endosymbiotic origin of mitochondria. In this report we describe the presence of a gene in Trichomonas vaginalis specifically related to mitochondrial chaperonin 60 (cpn60). A recent study indicates that a protein immunologically related to cpn60 is located in trichomonad hydrogenosomes. Together, these data provide evidence that ancestors of trichomonads perhaps harbored the endosymbiotic progenitors of mitochondria, but that these evolved into hydrogenosomes early in trichomonad evolution.

Architectural limits on split genes

Exon/intron architecture varies across the eukaryotic kingdom with large introns and small exons the rule in vertebrates and the opposite in lower eukaryotes. To investigate the relationship between exon and intron size in pre-mRNA processing, internally expanded exons were placed in vertebrate genes with small and large introns. Both exon and intron size influenced splicing phenotype. Intron size dictated if large exons were efficiently recognized. When introns were large, large exons were skipped; when introns were small, the same large exons were included. Thus, large exons were incompatible for splicing if and only if they were flanked by large introns. Both intron and exon size became problematic at ≈500 nt, although both exon and intron sequence influenced the size at which exons and introns failed to be recognized. These results indicate that present-day gene architecture reflects at least in part limitations on exon recognition. Furthermore, these results strengthen models that invoke pairing of splice sites during recognition of pre-mRNAs, and suggest that vertebrate consensus sequences support pairing across either introns or exons.

Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination

Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, to plant cells where it integrates into the nuclear genome via illegitimate recombination. Integration of the T-DNA results in small deletions of the plant target DNA, and may lead to truncation of the T-DNA borders and the production of filler DNA. We showed previously that T-DNA can also be transferred from A. tumefaciens to Saccharomyces cerevisiae and integrates into the yeast genome via homologous recombination. We show here that when the T-DNA lacks homology with the S. cerevisiae genome, it integrates at random positions via illegitimate recombination. From 11 lines the integrated T-DNA was cloned back to Escherichia coli along with yeast flanking sequences. The T-DNA borders and yeast DNA flanking the T-DNA were sequenced and characterized. It was found that T-DNA integration had resulted in target DNA deletions and sometimes T-DNA truncations or filler DNA formation. Therefore, the molecular mechanism of illegitimate recombination by which T-DNA integrates in higher and lower eukaryotes seems conserved.

Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I

Current evidence suggests that the length of poly(A) tails of bacterial mRNAs result from a competition between poly(A) polymerase and exoribonucleases that attack the 3′ ends of RNAs. Here, we show that host factor Hfq is also involved in poly(A) tail metabolism. Inactivation of the hfq gene reduces the length of poly(A) tails synthesized at the 3′ end of the rpsO mRNA by poly(A) polymerase I in vivo. In vitro, Hfq stimulates synthesis of long tails by poly(A) polymerase I. The strong binding of Hfq to oligoadenylated RNA probably explains why it stimulates elongation of primers that already harbor tails of 20–35 A. Polyadenylation becomes processive in the presence of Hfq. The similar properties of Hfq and the PABPII poly(A) binding protein, which stimulates poly(A) tail elongation in mammals, indicates that similar mechanisms control poly(A) tail synthesis in prokaryotes and eukaryotes.

A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase

The minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. Thus far, all eukaryotes have been shown to contain six highly related MCMs that apparently function together in DNA replication. Sequencing of the entire genome of the thermophilic archaeon Methanobacterium thermoautotrophicum has allowed us to identify only a single MCM-like gene (ORF Mt1770). This gene is most similar to MCM4 in eukaryotic cells. Here we have expressed and purified the M. thermoautotrophicum MCM protein. The purified protein forms a complex that has a molecular mass of ≈850 kDa, consistent with formation of a double hexamer. The protein has an ATP-independent DNA-binding activity, a DNA-stimulated ATPase activity that discriminates between single- and double-stranded DNA, and a strand-displacement (helicase) activity that can unwind up to 500 base pairs. The 3′ to 5′ helicase activity requires both ATP hydrolysis and a functional nucleotide-binding site. Moreover, the double hexamer form is the active helicase. It is therefore likely that an MCM complex acts as the replicative DNA helicase in eukaryotes and archaea. The simplified replication machinery in archaea may provide a simplified model for assembly of the machinery required for initiation of eukaryotic DNA replication.

An electric lobe suppressor for a yeast choline transport mutation belongs to a new family of transporter-like proteins

Choline is an important metabolite in all cells due to the major contribution of phosphatidylcholine to the production of membranes, but it takes on an added role in cholinergic neurons where it participates in the synthesis of the neurotransmitter acetylcholine. We have cloned a suppressor for a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation. The full-length clone encodes a protein with 10 putative transmembrane domains, two of which contain transporter-like motifs, and whose expression increased high-affinity choline uptake in mutant yeast. The gene was called CTL1 for its choline transporter-like properties. The homologous rat gene, rCTL1, was isolated and found to be highly expressed as a 3.5-kb transcript in the spinal cord and brain and as a 5-kb transcript in the colon. In situ hybridization showed strong expression of rCTL1 in motor neurons and oligodendrocytes and to a lesser extent in various neuronal populations throughout the rat brain. High levels of rCTL1 were also identified in the mucosal cell layer of the colon. Although the sequence of the CTL1 gene shows clear homology with a single gene in Caenorhabditis elegans, several homologous genes are found in mammals (CTL2–4). These results establish a new family of genes for transporter-like proteins in eukaryotes and suggest that one of its members, CTL1, is involved in supplying choline to certain cell types, including a specific subset of cholinergic neurons.

A novel precursor recognition element facilitates posttranslational binding to the signal recognition particle in chloroplasts

Signal recognition particles (SRPs) in the cytosols of prokaryotes and eukaryotes are used to target proteins to cytoplasmic membranes and the endoplasmic reticulum, respectively. The mechanism of targeting relies on cotranslational SRP binding to hydrophobic signal sequences. An organellar SRP identified in chloroplasts (cpSRP) is unusual in that it functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins. In assays that reconstitute thylakoid integration of the light harvesting chlorophyll-binding protein (LHCP), stromal cpSRP binds LHCP posttranslationally to form a cpSRP/LHCP transit complex, which is believed to represent the LHCP form targeted to thylakoids. In this investigation, we have identified an 18-aa sequence motif in LHCP (L18) that, along with a hydrophobic domain, is required for transit complex formation. Fusion of L18 to the amino terminus of an endoplasmic reticulum-targeted protein, preprolactin, led to transit complex formation whereas wild-type preprolactin exhibited no ability to form a transit complex. In addition, a synthetic L18 peptide, which competed with LHCP for transit complex formation, caused a parallel inhibition of LHCP integration. Translocation of proteins by the thylakoid Sec and Delta pH transport systems was unaffected by the highest concentration of L18 peptide examined. Our data indicate that a motif contained in L18 functions in precursor recruitment to the posttranslational SRP pathway, one of at least four different thylakoid sorting pathways used by chloroplasts.