The plastid aldolase gene fromChlamydomonas reinhardtii: Intron/exon organization, evolution, and promoter structure

Genomic clones encoding the plastidic fructose- 1,6-bisphosphate aldolase ofChlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of theC. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5 border sequences of introns in the aldolase gene ofC. reinhardtii exhibit the conserved plant consensus sequence. The 3 acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in theC. reinhardtii genome.     

Sequence of the region downstream of theVitreoscilla hemoglobin gene:vgb is not part of a multigene operon

The 1668 base pairs (bp) downstream of theVitreoscilla hemoglobin gene were sequenced in the hope of finding related genes that might be part of an operon. Instead, a sequence was found that constituted an open reading frame (ORF) of 569 amino acids (apparently the carboxy-terminal part of a larger ORF), in the direction opposite to the hemoglobin gene. This sequence was found to have 64% similarity with the 1685 by at the 3 end of theEscherichia coli uvrA gene. The inferred amino acid sequence of the Vitreoscilla DNA has 69% similarity with the corresponding sequence of theE. coli uvrA protein, with similarities of 90, 100, and 85% in the helix-turn-helix, C-terminal ATP binding, and C-terminal zinc finger domains, respectively. The distance between the 3 ends of theVitreoscilla hemoglobin anduvrA genes is 63 bp.     

Reactivity of cysteinyl, arginyl, and lysyl residues ofEscherichia coli phosphoenolpyruvate carboxykinase against group-specific chemical reagents

Calcium-activated phosphoenolpyruvate carboxykinase fromEscheria coli is not inactivated by a number of sulfhydryl-directed reagents [5,5-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N-(5-sulfo-l-naphthylethylenediamine)], unlike phosphoenolpyruvate carboxykinase from other organisms. On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal. The substrates, ADP plus PEP in the presence of Mn²⁺, protect the enzyme against inactivation by the diones. Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme. Chemical modification by pyridoxal 5-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine. The inactivation by this reagent is also prevented by the substrates. Binding stoichiometries of 1.25 and 0.30mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively. The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.Abbreviations used: DTNB, 5,5-dithiobis(2-nitrobenzoate); Hepes, N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid; 1,5-IAEDANS, N-(iodoacetyl)-N-(5-sulfo-1-naphthyl) ethylenediamine; EPE, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PG, 1-pyrenylglyoxal; PLP, pyridoxal 5-phosphate.     

The Vitreoscilla hemoglobin gene: Molecular cloning, nucleotide sequence and genetic expression in Escherichia coli

Summary Vitreoscilla hemoglobin is involved in oxygen metabolism of this bacterium, possibly in an unusual role for a microbe. We have isolated the Vitreoscilla hemoglobin structural gene from a pUC19 genomic library using mixed oligodeoxy-nucleotide probes based on the reported amino acid sequence of the protein. The gene is expressed in Escherichia coli from its natural promoter as a major cellular protein. The nucleotide sequence, which is in complete agrecment with the known amino acid sequence of the protein, suggests the existence of promoter and ribosome binding sites with a high degree of homology to consensus E. coli upstream sequences. In the case of at least some amino acids, a codon usage bias can be detected which is different from the biased codon usage pattern in E. coli. The down-stream sequence exhibits homology with the 3 end sequences of several plant leghemoglobin genes. E. coli cells expressing the gene contain greater than fivefold more heme than controls.     

Sequence analysis of linked maize 22 kDa α-zein genes

We have determined the nucleotide sequence of a 7343 bp zein genomic clone (gZ22.8H3) from the maize inbred W64A. Computer-aided analysis of the DNA sequence revealed two contiguous 22 kDa -zein genes. The 5 gene (gZ22.8) encodes a complete polypeptide and contains putative regulatory sequences in both the 5 and 3 flanking regions that are typical of zein genes. In contrast, the 3 gene (gZ22.8) appears to be a pseudogene, because it contains numerous insertions and deletions that would prevent translation of the mRNA. Alignment of the 5 and 3 flanking sequences of both genes indicated that they resulted from a 3.3 kb DNA duplication event.     

Conservation of the 3′-untranslated regions of calmodulin mRNAs in cetaceans

Three separate calmodulin (CaM) genes (I, II and III) encoding an identical CaM protein but differing in the 5- and 3-untranslated regions of each of the three mRNAs are present and highly conserved in all mammals (so far examined). Primers complementary to the 3- untranslated region (3UTR) of each of the three mRNAs occurring in human, rat and mouse were synthesized and used to amplify regions of the 3UTR from genomic DNA isolated from cetaceans, specifically from the bottled-nosed dolphin (Tursiops truncates), the pygmy sperm whale (Kogia breviceps) and the humpback whale (Megaptera novaeangliae). Using several primers and PCR conditions, the three CaM genes were identified in all three species by this method with one exception. The sequenced regions of the 3UTRs of the three genes of the cetaceans exhibited a high percentage identity when compared to the corresponding regions of these three CaM mRNAs isolated from humans (85-96%). These partial sequences of the 3UTR regions and the corresponding regions for humans, rats and mice that were available from the database were aligned and a phylogenetic tree was constructed. The three CaM genes from all species showed a close phylogenetic relationship based on these 3UTR sequences. Such high conservation of the 3UTRs suggests a specialized and significant function for this region in mammals.     

Tissue-specific and developmental regulation of a gene encoding a low molecular weight sulfur-rich protein in soybean seeds

A gene corresponding to a cDNA clone, SE60, encoding a low molecular weight sulfur-rich protein in soybean seeds was isolated from a soybean genomic library and characterized at the nucleotide level. The SE60 gene is interrupted by an intervening sequence of 694 by in size. The 5 flanking region of the gene contained various regulatory sequences such as the RY repeat and CACA elements found in other seed protein genes of legumes. The SE60 gene encoded a pre-protein of 75 amino acids, having a signal sequence of 28 amino acids at the N-terminus. The mature protein of 47 amino acids was basic and cysteine-rich. Northern blot analysis suggested that the SE60 gene is expressed in a tissue-specific and developmentally regulated manner during soybean seed development. The SE60 genes form a small multigene family composed of about four members in the soybean genome.     

ThePenicillium chrysogenum andAspergillus nidulans wetA developmental regulatory genes are functionally equivalent

Aspergillus nidulans andPenicillium chrysogenum are related fungi that reproduce asexually by forming multicellular conidiophores and uninucleate conidia. InA. nidulans, spore maturation is controlled by thewetA (AwetA) regulatory gene. We cloned a homologous gene (PwetA) fromP. chrysogenum to determine if spore maturation is regulated by a similar mechanism in this species. ThePwetA andAwetA genes are similar in structure and functional organization. The inferred polypeptides share 77% overall amino acid sequence similarity, with several regions having > 85% similarity. The genes also had significant, local sequence similarities in their 5 flanking regions, including conserved binding sites for the product of the regulatory geneabaA.PwetA fully complemented anA. nidulans wetA deletion mutation, demonstrating thatPwetA and its 5 regulatory sequences function normally inA. nidulans. These results indicate that the mechanisms controlling sporulation inA. nidulans andP. chrysogenum are evolutionarily conserved.     

A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants

Summary We have isolated a novel gene, denoted USP, from Vicia faba var. minor, which corresponds to the most abundant mRNA present in cotyledons during early seed development; however, the corresponding protein does not accumulate in cotyledons. The characterized USP gene with its two introns is 1 of about 15 members of a gene family. A fragment comprising 637 bp of 5 flanking sequence and the total 5 untranslated region was shown to be sufficient to drive the mainly seed-specific expression of two reporter genes, coding for neomycin phosphotransferase 11 and -glucuronidase, in transgenic Arabidopsis thaliana and Nicotiana tabacum plants. We showed that the USP promoter becomes active in transgenic tobacco seeds in both the embryo and the endosperm, whereas its activity in Arabidopsis is detectable only in the embryo. Moreover, we demonstrated a transient activity pattern of the USP promoter in root tips of both transgenic host species.     

Identification of a multigene family for small heat shock proteins in soybean and physical characterization of one individual gene coding region

When soybean seedlings are tranferred from 28 to 40 ° C, a heat shock (hs) response is elicited. This is characterized by the synthesis of a new set of proteins (hs-proteins) and by cessation of normal protein synthesis (8). At the level of poly(A)mRNA, a new class of highly abundant RNAs appears which encodes a group of hs-proteins in the low molecular weight range of 15–18 kD (11). The classification of these proteins/genes into several sub-classes is based on a complex sequence relationship for class I protein/genes.This was confirmed by both the complexity and the similarity of southern blot hybridization patterns of genomic DNA digests with class I cDNA-probes. Genomic DNA clones (obtained from -libraries by screening with cDNA-probes) for the class I gene 1968 showed cross hybridization with all other class I cDNA-probes. Higher specificity of gene/protein correlation was obtained by variation of hybridization criteria. The specificity of cDNA clone 1968 for the genomic DNA clone hs68-7 was demonstrated by thermal stability of hybridization at 55 ° C and 65 ° C in 50% formamide compared to other cross-reacting probes. The correlation of clone 1968 with a specific hs-protein was obtained by temperature dependent release of hybrid selected hs-mRNAs at 50, 60, 70 and 85 ° C followed byin vitro translation and two-dimensional gel analysis. The coding regions of hs-genes on genomic DNA clones were mapped by R-loop formation. The position of R-loops was mapped relative to certain restriction sites on subclones of hs68-7 DNA. The polarity of hs-genes was determined by attaching X174RF-DNA labels to the 3 poly(A)-tails of the mRNAs of R-loops.     

Mapping of human thyroglobulin gene on the long arm of chromosome 8 by in situ hybridization

Summary We report the structural organization of a segment of the human thyroglobulin gene, located 70kb from the 3 end of the gene, containing the exons 8 and 9 starting from the 3 end. Selected probes from this region have been used for the chromosomal mapping of the thyroglobulin gene by in situ hybridization techniques. Only one site in the human haploid karyotype is labeled with the genomic DNA probes. Twenty percent of the grains are localized on the long arm of chromosome 8, mostly in the subregion q-2-23 q-2-24 of the long arm of chromosome 8. The localization of the autoradiographic grains suggests a subregional assignment of the human thyroglobulin gene locus to 8q 2–23 or 8q 2–24.     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

Multicopy plasmid suppression of stationary phase chaperone toxicity in Escherichia coli by phosphogluconate dehydratase and the N-terminus of DnaK

Overproduction of DnaK in Escherichia coli results in a bacteriocidal effect. This effect is most acute in stationary phase cells. A selection scheme was developed to isolate multicopy suppressors from an E. coli plasmid expression library, which overcome the stationary phase toxicity of excess DnaK. Two suppressor plasmids were recovered which contained inserts of 1.85 kb and 2.69 kb, respectively. Rearranged and deleted plasmid derivatives were constructed and used to further localize the suppressors. DNA sequence analysis demonstrated that one suppressor encoded phosphogluconate dehydratase (Edd) while the other suppressor encoded the N-terminal 237 amino acids of DnaK itself (DnaK). Strains bearing the suppressor plasmids constitutively overproduced proteins with apparent masses of 66 kDa (Edd) and 37 kDa (DnaK) as determined by gel electrophoresis. Western blot analysis using polyclonal antisera specific for either Edd or DnaK confirmed the identity of these overproduced proteins. Suppression of DnaK toxicity was eliminated by the introduction of a + 1 frameshift mutation early in the respective coding regions of either of the two suppressors. These results suggest that suppressor gene translation plays a role in the mechanism of DnaK suppression.     

Partial conservation of the 5′ ndhE-psaC-ndhD 3′ gene arrangement of chloroplasts in the cyanobacterium Synechocystis sp. PCC 6803: implications for NDH-D function in cyanobacteria and chloroplasts

The psaC gene, which encodes the 8.9 kDa iron-sulfur containing subunit of Photosystem I, has been sequenced from Synechocystis sp. PCC 6803 and shows greater similarity to reported plant sequences than other cyanobacterial psaC sequences. The deduced amino acid sequence of the protein encoded by the Synechocystis psaC gene is identical to the tobacco PSA-C sequence. In plants psaC is located in the small single-copy region of the chloroplast genome between two genes (designated ndhE and ndhD) with similarity to genes encoding subunits of the mitochondrial NADH Dehydrogenase Complex I. The 5 ndhE-psaC-ndhD3 gene arrangement of higher plants is only partially conserved in Synechocystis. An open reading frame (ORF) upstream of the Synechocystis psaC gene has 85% identity to the tobacco ndhE gene. Downstream of psaC there is a 273 bp ORF with 48% identity to the 5 portion of the tobacco ndhD gene (1527 bp). psaC, ndhE and the region of similarity to ndhD are present in a single copy in the Synechocystis genome. Part of the wheat ndhD gene was sequenced and used as a probe for the presence of the 3 portion of the ndhD gene. The wheat ndhD probe did not hybridize to Synechocystis or Anabaena sp. PCC 7120 genomic DNA, but did hybridize to Oenothera chloroplast DNA. These results indicate the complete ndhD gene is absent in two cyanobacteria, and raises the question of what role, if any, the ndhD gene product plays in the facultative heterotroph Synechocystis sp. PCC 6803.     

Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis

Summary The ptsG gene of Bacillus subtilis encodes Enzyme II^G1c of the phosphoenolpyruvate: glucose phosphotransferase system. The 3 end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes III^Glc of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5 end. These results, combined with the sequence of the 3 end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes II^Glc and II^Nag of E. coli but which are present in the II^G1c-like protein encoded by the E. coli malX gene.     

A single-base deletion in soybean flavonoid 3′-hydroxylase gene is associated with gray pubescence color

The T locus of soybean (Glycine max (L.) Merr.) controls pubescence and seed coat color and is presumed to encode flavonoid 3-hydroxylase (F3H). The dominant T and the recessive t allele of the locus produce brown and gray pubescence, respectively. PCR primers were constructed based on the sequence of a soybean EST clone homologous to the F3H gene. A putative full-length cDNA, sf3h1 was isolated by 3 and 5 RACE. Sequence analysis revealed that sf3h1 consists of 1690 nucleotides encoding 513 amino acids. It had 68% and 66% homology with corresponding F3H protein sequences of petunia and Arabidopsis, respectively. A conserved amino acid sequence of F3H proteins, GGEK, was found in the deduced polypeptide. Sequence analysis of the gene from a pair of near-isogenic lines for T, To7B (TT, brown) and To7G (tt, gray) revealed that they differed by a single C deletion in the coding region of To7G. The deletion changed the subsequent reading frame resulting in a truncated polypeptide lacking the GGEK consensus sequence and the heme-binding domain. Genomic Southern analysis probed by sf3h1 revealed restriction fragment length polymorphisms between cultivars with different pubescence color. Further, sf3h1 was mapped at the same position with T locus on LG3(c2). PCR-RFLP analysis was performed to detect the single-base deletion. To7B and three cultivars with brown pubescence exhibited shorter fragments, while To7G and three cultivars with gray pubescence had longer fragments due to the single-base deletion. The PCR-RFLP marker co-segregated with genotypes at the Tlocus in a F₂ population segregating for the T locus. The above results strongly suggest that sf3h1 represents the T gene of soybean responsible for pubescence color and that the single-base deletion may be responsible for gray pubescence color.     

Characterization of a Gy4 glycinin gene from soybean Glycine max cv. Forrest

The glycinin gene family encoding the glycinin subunits in soybean plants is composed of at least five gene members. A genomic clone S312 containing the Gy4 gene from a genomic library of cv. Forrest was isolated and partially characterized. The organization of this gene was found to be similar to that of a null allele from cv. Raiden, but different from the Gy4 gene from cv. Dare. The complete nucleotide sequence of this gene has been determined. It is 2599 bp long consisting of four exons and three introns. Comparing the DNA sequences between this gene and the gene from Dare and a null allele from Raiden, the difference found in the coding region was 5-GCAGTGCAAG-3 (nt 824 to 833) in the former case versus 5-TGGAGTTGCAATT-3 (nt 1314 to 1326) in the latter case in the exon 2 domain, resulting in three amino acid differences and one amino acid absence. Some other differences were also found in the non-coding region. The coding sequence and 5-flanking region of the Gy4 gene, when compared with that of other legumin genes as well as group 1 glycinin subunit genes, revealed some interesting features: (1) a transposable element-like sequence was found in the hypervariable region (HVR) of the exon 3 domain, which was lacking in the legumin and the glycinin group 1 genes; (2) in the 5-flanking region from nt –145 to –1, two high-homology sequences were found: one from nt –141 to nt –132, the other from nt –118 to nt –92 which includes the legumin box and the RY repeat element.     

The Lotus japonicus LjNOD70 nodulin gene encodes a protein with similarities to transporters

A novel nodule-specific gene, LjNOD70, associated with late stages in Lotus japonicus nodule development and/or functioning was characterized. The LjNOD70 gene is a member of a small family of closely related L. japonicus genes. Two major mRNA species corresponding to the LjNOD70 gene were identified in nodules and shown to be the result of a mechanism resembling alternative splicing. The longer, presumably unspliced, mRNA species was shown to contain a single open reading frame (ORF), encoding a polytopic hydrophobic protein, LjN70, with a predicted molecular mass of 70 kDa. The second, presumably spliced, mRNA species was shown to be less abundant in nodules. The absence of the presumptive intron was found to divide the reading frame into an upstream and a downstream ORF encoding the partial N- and C-terminal regions of the LjN70 protein, respectively. The predicted amino acid sequence of nodulin LjN70 revealed structural features characteristic of transport proteins, and was found to share similarity with the oxalate/formate exchange protein of Oxalobacter formigenes. Therefore, we postulate that the L. japonicus LjNOD70 gene family encodes nodule-specific transport proteins, which may have evolved as a result of exon-intron shuffling.     

Characterization of fibroblast cytoplasmic proteins that bind to the 3′ UTR of human catalse mRNA

The excessive expression of catalase protein and its activity in cultured skin fibroblast from Zellweger Syndrome (ZS), a disorder of peroxisomal biogenesis, was found to be regulated at the translational level (J. Neurochem. 67: 2373-2378, 1996). Overall there is a considerable increase in the association of catalase mRNA with polysomes in ZS cell lines as compared to control indicating translational upregulation. To investigate the possibility that RNA-protein interactions are involved in the mediation of this increase in translation, the interaction between 3 untranslated region of human catalase mRNA and human fibroblast cytoplasmic proteins were investigated by RNA gel shift assay technique. Competition experiments demonstrated that all the 600 bases of 3 UTR (of human catalase gene) was required for efficient binding. Catalase RNA- protein interaction was sensitive to the altered redox state in these in vitro assays and this RNA-protein interaction could be enhanced by the addition of -mercaptoethanol in cytoplasm from control fibroblast but not in cytoplasm from ZS fibroblast. UV cross linked RNA-protein complexes on SDS polyacrylamide gel electrophoresis revealed the presence of at least four protein bands with approximate molecular masses of 38 kDa, 50 kDa, 66 kDa and 80 kDa. The potential role of these mRNA binding proteins in the regulation of catalase gene expression is discussed.