Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster

An extensive, highly diversified multigene family of novel immune-type receptor (nitr) genes has been defined in Danio rerio (zebrafish). The genes are predicted to encode type I transmembrane glycoproteins consisting of extracellular variable (V) and V-like C2 (V/C2) domains, a transmembrane region and a cytoplasmic tail. All of the genes examined encode immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. Radiation hybrid panel mapping and analysis of a deletion mutant line (b240) indicate that a minimum of ≈40 nitr genes are contiguous in the genome and span ≈0.6 Mb near the top of zebrafish linkage group 7. One flanking region of the nitr gene complex shares conserved synteny with a region of mouse chromosome 7, which shares conserved synteny with human 19q13.3-q13.4 that encodes the leukocyte receptor cluster. Antibody-induced crosslinking of Nitrs that have been introduced into a human natural killer cell line inhibits the phosphorylation of mitogen-activated protein kinase that is triggered by natural killer-sensitive tumor target cells. Nitrs likely represent intermediates in the evolution of the leukocyte receptor cluster.

Identification of Two Type V Myosins in Fission Yeast, One of Which Functions in Polarized Cell Growth and Moves Rapidly in the Cell

We characterized the novel Schizosaccharomyces pombe genes myo4+ and myo5+, both of which encode myosin-V heavy chains. Disruption of myo4 caused a defect in cell growth and led to an abnormal accumulation of secretory vesicles throughout the cytoplasm. The mutant cells were rounder than normal, although the sites for cell polarization were still established. Elongation of the cell ends and completion of septation required more time than in wild-type cells, indicating that Myo4 functions in polarized growth both at the cell ends and during septation. Consistent with this conclusion, Myo4 was localized around the growing cell ends, the medial F-actin ring, and the septum as a cluster of dot structures. In living cells, the dots of green fluorescent protein-tagged Myo4 moved rapidly around these regions. The localization and movement of Myo4 were dependent on both F-actin cables and its motor activity but seemed to be independent of microtubules. Moreover, the motor activity of Myo4 was essential for its function. These results suggest that Myo4 is involved in polarized cell growth by moving with a secretory vesicle along the F-actin cables around the sites for polarization. In contrast, the phenotype of myo5 null cells was indistinguishable from that of wild-type cells. This and other data suggest that Myo5 has a role distinct from that of Myo4.

Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila

Sequence comparisons of genomes or expressed sequence tags (ESTs) from related organisms provide insight into functional conservation and diversification. We compare the sequences of ESTs from the male accessory gland of Drosophila simulans to their orthologs in its close relative Drosophila melanogaster, and demonstrate rapid divergence of many of these reproductive genes. Nineteen (∼11%) of 176 independent genes identified in the EST screen contain protein-coding regions with an excess of nonsynonymous over synonymous changes, suggesting that their divergence has been accelerated by positive Darwinian selection. Genes that encode putative accessory gland-specific seminal fluid proteins had a significantly elevated level of nonsynonymous substitution relative to nonaccessory gland-specific genes. With the 57 new accessory gland genes reported here, we predict that ∼90% of the male accessory gland genes have been identified. The evolutionary EST approach applied here to identify putative targets of adaptive evolution is readily applicable to other tissues and organisms.

A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans

The Deleted in AZoospermia (DAZ) genes encode potential RNA-binding proteins that are expressed exclusively in prenatal and postnatal germ cells and are strong candidates for human fertility factors. Here we report the identification of an additional member of the DAZ gene family, which we have called BOULE. With the identification of this gene, it is clear that the human DAZ gene family contains at least three members: DAZ, a Y-chromosome gene cluster that arose 30–40 million years ago and whose deletion is linked to infertility in men; DAZL, the “father” of DAZ, a gene that maps to human chromosome 3 and has homologs required for both female and male germ cell development in other organisms; and BOULE, a gene that we propose is the “grandfather” of DAZ and maps to human chromosome 2. Human and mouse BOULE resemble the invertebrate meiotic regulator Boule, the proposed ortholog of DAZ, in sequence and expression pattern and hence likely perform a similar meiotic function. In contrast, the previously identified human DAZ and DAZL are expressed much earlier than BOULE in prenatal germ stem cells and spermatogonia; DAZL also is expressed in female germ cells. These data suggest that homologs of the DAZ gene family can be grouped into two subfamilies (BOULE and DAZL) and that members of the DAZ family evolved from an ancestral meiotic regulator, Boule, to assume distinct, yet overlapping, functions in germ cell development.

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: Population genetics, human serologic response, and gene transcription

Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase–PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research.

The First Step of Gibberellin Biosynthesis in Pumpkin Is Catalyzed by at Least Two Copalyl Diphosphate Synthases Encoded by Differentially Regulated Genes

The first step in gibberellin biosynthesis is catalyzed by copalyl diphosphate synthase (CPS) and ent-kaurene synthase. We have cloned from pumpkin (Cucurbita maxima L.) two cDNAs, CmCPS1 and CmCPS2, that each encode a CPS. Both recombinant fusion CmCPS proteins were active in vitro. CPS are translocated into plastids and processed by cleavage of transit peptides. For CmCPS1 and CmCPS2, the putative transit peptides cannot exceed the first 99 and 107 amino acids, respectively, because longer N-terminal deletions abolished activity. Levels of both CmCPS transcripts were strictly regulated in an organ-specific and developmental manner. Both transcripts were almost undetectable in leaves and were abundant in petioles. CmCPS1 transcript levels were high in young cotyledons and low in roots. In contrast, CmCPS2 transcripts were undetectable in cotyledons but present at significant levels in roots. In hypocotyls, apices, and petioles, CmCPS1 transcript levels decreased with age much more rapidly than those of CmCPS2. We speculate that CmCPS1 expression is correlated with the early stages of organ development, whereas CmCPS2 expression is correlated with subsequent growth. In contrast, C. maxima ent-kaurene synthase transcripts were detected in every organ at almost constant levels. Thus, ent-kaurene biosynthesis may be regulated through control of CPS expression.

Phytochrome Regulates Gibberellin Biosynthesis during Germination of Photoblastic Lettuce Seeds1

Germination of lettuce (Lactuca sativa L.) seed is regulated by phytochrome. The requirement for red light is circumvented by the application of gibberellin (GA). We have previously shown that the endogenous content of GA1, the main bioactive GA in lettuce seeds, increases after red-light treatment. To clarify which step of GA1 synthesis is regulated by phytochrome, cDNAs encoding GA 20-oxidases (Ls20ox1 and Ls20ox2, for L. sativa GA 20-oxidase) and 3β-hydroxylases (Ls3h1 and Ls3h2 for L. sativa GA 3β-hydroxylase) were isolated from lettuce seeds by reverse-transcription polymerase chain reaction. Functional analysis of recombinant proteins expressed in Escherichia coli confirmed that the Ls20ox and Ls3h encode GA 20-oxidases and 3β-hydroxylases, respectively. Northern-blot analysis showed that Ls3h1 expression was dramatically induced by red-light treatment within 2 h, and that this effect was canceled by a subsequent far-red-light treatment. Ls3h2 mRNA was not detected in seeds that had been allowed to imbibe under any light conditions. Expression of the two Ls20ox genes was induced by initial imbibition alone in the dark. The level of Ls20ox2 mRNA decreased after the red-light treatment, whereas that of Ls20ox1 was unaffected by light. These results suggest that red light promotes GA1 synthesis in lettuce seeds by inducing Ls3h1 expression via phytochrome action.

Systematic Reverse Genetics of Transfer-DNA-Tagged Lines of Arabidopsis1 : Isolation of Mutations in the Cytochrome P450 Gene Superfamily

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.

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.

RNA Polymerase Subunits Encoded by the Plastid rpo Genes Are Not Shared with the Nucleus-Encoded Plastid Enzyme1

Plastid genes in photosynthetic higher plants are transcribed by at least two RNA polymerases. The plastid rpoA, rpoB, rpoC1, and rpoC2 genes encode subunits of the plastid-encoded plastid RNA polymerase (PEP), an Escherichia coli-like core enzyme. The second enzyme is referred to as the nucleus-encoded plastid RNA polymerase (NEP), since its subunits are assumed to be encoded in the nucleus. Promoters for NEP have been previously characterized in tobacco plants lacking PEP due to targeted deletion of rpoB (encoding the β-subunit) from the plastid genome. To determine if NEP and PEP share any essential subunits, the rpoA, rpoC1, and rpoC2 genes encoding the PEP α-, β′-, and β"-subunits were removed by targeted gene deletion from the plastid genome. We report here that deletion of each of these genes yielded photosynthetically defective plants that lack PEP activity while maintaining transcription specificity from NEP promoters. Therefore, rpoA, rpoB, rpoC1, and rpoC2 encode PEP subunits that are not essential components of the NEP transcription machinery. Furthermore, our data indicate that no functional copy of rpoA, rpoB, rpoC1, or rpoC2 that could complement the deleted plastid rpo genes exists outside the plastids.

Gibberellin Dose-Response Regulation of GA4 Gene Transcript Levels in Arabidopsis1

The gibberellins (GAs) are a complex family of diterpenoid compounds, some of which are potent endogenous regulators of plant growth. As part of a feedback control of endogenous GA levels, active GAs negatively regulate the abundance of mRNA transcripts encoding GA biosynthesis enzymes. For example, Arabidopsis GA4 gene transcripts encode GA 3β-hydroxylase, an enzyme that catalyzes the conversion of inactive to active GAs. Here we show that active GAs regulate GA4 transcript abundance in a dose-dependent manner, and that down-regulation of GA4 transcript abundance is effected by GA4 (the product of 3β-hydroxylation) but not by its immediate precursor GA9 (the substrate). Comparison of several different GA structures showed that GAs active in promoting hypocotyl elongation were also active in regulating GA4 transcript abundance, suggesting that similar GA:receptor and subsequent signal transduction processes control these two responses. It is interesting that these activities were not restricted to 3β-hydroxylated GAs, being also exhibited by structures that were not 3β-hydroxylated but that had another electronegative group at C-3. We also show that GA-mediated control of GA4 transcript abundance is disrupted in the GA-response mutants gai and spy-5. These observations define a sensitive homeostatic mechanism whereby plants may regulate their endogenous GA levels.

Characterization of Two Glutamate Decarboxylase cDNA Clones from Arabidopsis

Two distinct cDNA clones encoding for the glutamate decarboxylase (GAD) isoenzymes GAD1 and GAD2 from Arabidopsis (L.) Heynh. were characterized. The open reading frames for GAD1 and GAD2 were expressed in Escherichia coli and the recombinant proteins were purified by affinity chromatography. Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1 and GAD2 encode for 58- and 56-kD peptides, respectively. The enzymatic activities of the pure recombinant GAD1 and GAD2 proteins were stimulated 35- and 13-fold, respectively, by Ca2+/calmodulin but not by Ca2+ or calmodulin alone. Southern-blot analysis of genomic DNA suggests that there is only one copy of each gene in Arabidopsis. The GAD1 transcript and a corresponding 58-kD peptide were detected in roots only. Conversely, the GAD2 transcript and a corresponding 56-kD peptide were detected in all organs tested. The specific activity, GAD2 transcript, and 56-kD peptide increased in leaves of plants treated with 10 mm NH4Cl, 5 mm NH4NO3, 5 mm glutamic acid, or 5 mm glutamine as the sole nitrogen source compared with samples from plants treated with 10 mm KNO3. The results from these experiments suggest that in leaves GAD activity is partially controlled by gene expression or RNA stability. Results from preliminary analyses of different tissues imply that these tendencies were not the same in flower stalks and flowers, suggesting that other factors may control GAD activity in these organs. The results from this investigation demonstrate that GAD activity in leaves is altered by different nitrogen treatments, suggesting that GAD2 may play a unique role in nitrogen metabolism.

Dedicated Roles of Plastid Transketolases during the Early Onset of Isoprenoid Biogenesis in Pepper Fruits1

Isopentenyl diphosphate (IPP), which is produced from mevalonic acid or other nonmevalonic substrates, is the universal precursor of isoprenoids in nature. Despite the presence of several isoprenoid compounds in plastids, enzymes of the mevalonate pathway leading to IPP formation have never been isolated or identified to our knowledge. We now describe the characterization of two pepper (Capsicum annuum L.) cDNAs, CapTKT1 and CapTKT2, that encode transketolases having distinct and dedicated specificities. CapTKT1 is primarily involved in plastidial pentose phosphate and glycolytic cycle integration, whereas CapTKT2 initiates the synthesis of isoprenoids in plastids via the nonmevalonic acid pathway. From pyruvate and glyceraldehyde-3-phosphate, CapTKT2 catalyzes the formation of 1-deoxy-xylulose-5-phosphate, the IPP precursor. CapTKT1 is almost constitutively expressed during the chloroplast-to-chromoplast transition, whereas CapTKT2 is overexpressed during this period, probably to furnish the IPP necessary for increased carotenoid biosynthesis. Because deoxy-xylulose phosphate is shared by the plastid pathways of isoprenoid, thiamine (vitamin B1), and pyridoxine (vitamin B6) biosynthesis, our results may explain why albino phenotypes usually occur in thiamine-deficient plants.

Premature Polyadenylation at Multiple Sites within a Bacillus thuringiensis Toxin Gene-Coding Region1

Some foreign genes introduced into plants are poorly expressed, even when transcription is controlled by a strong promoter. Perhaps the best examples of this problem are the cry genes of Bacillus thuringiensis (B.t.), which encode the insecticidal proteins commonly referred to as B.t. toxins. As a step toward overcoming such problems most effectively, we sought to elucidate the mechanisms limiting the expression of a typical B.t.-toxin gene, cryIA(c), which accumulates very little mRNA in tobacco (Nicotiana tabacum) cells. Most cell lines transformed with the cryIA(c) B.t.-toxin gene accumulate short, polyadenylated transcripts. The abundance of these transcripts can be increased by treating the cells with cycloheximide, a translation inhibitor that can stabilize many unstable transcripts. Using a series of hybridizations, reverse-transcriptase polymerase chain reactions, and RNase-H-digestion experiments, poly(A+) addition sites were identified in the B.t.-toxin-coding region corresponding to the short transcripts. A fourth polyadenylation site was identified using a chimeric gene. These results demonstrate for the first time to our knowledge that premature polyadenylation can limit the expression of a foreign gene in plants. Moreover, this work emphasizes that further study of the fundamental principles governing polyadenylation in plants will have basic as well as applied significance.

Molecular and Biochemical Characterization of Cytosolic Phosphoglucomutase in Maize1 : Expression during Development and in Response to Oxygen Deprivation

Phosphoglucomutase (PGM) catalyzes the interconversion of glucose (Glc)-1- and Glc-6-phosphate in the synthesis and consumption of sucrose. We isolated two maize (Zea mays L.) cDNAs that encode PGM with 98.5% identity in their deduced amino acid sequence. Southern-blot analysis with genomic DNA from lines with different Pgm1 and Pgm2 genotypes suggested that the cDNAs encode the two known cytosolic PGM isozymes, PGM1 and PGM2. The cytosolic PGMs of maize are distinct from a plastidic PGM of spinach (Spinacia oleracea). The deduced amino acid sequences of the cytosolic PGMs contain the conserved phosphate-transfer catalytic center and the metal-ion-binding site of known prokaryotic and eukaryotic PGMs. PGM mRNA was detectable by RNA-blot analysis in all tissues and organs examined except silk. A reduction in PGM mRNA accumulation was detected in roots deprived of O2 for 24 h, along with reduced synthesis of a PGM identified as a 67-kD phosphoprotein on two-dimensional gels. Therefore, PGM is not one of the so-called “anaerobic polypeptides.” Nevertheless, the specific activity of PGM was not significantly affected in roots deprived of O2 for 24 h. We propose that PGM is a stable protein and that existing levels are sufficient to maintain the flux of Glc-1-phosphate into glycolysis under O2 deprivation.