Characterization of the 3′ untranslated region of mouse DNA topoisomerase IIα mRNA

Expression of DNA topoisomerase IIα protein varies through the cell cycle with its peak in G₂/M. This cell-cycle-dependent expression depends on changes in topoisomerase IIα mRNA stability as well as promoter activity. We isolated the 3′ genomic region of the mouse topoisomerase IIα gene and investigated whether or not the 3′ untranslated region (UTR) of the topoisomerase IIα mRNA participates in the cell-cycle-dependent mRNA stability. Interestingly, genomic- and RT-PCR analyses revealed that the topoisomerase IIα 3′ UTR is formed via splicing in mouse, but not in human and hamster. Comparison of the mouse 3′ region with the human and hamster regions suggests that this mouse-specific splicing has resulted from an accidental acquisition of the consensus 5′ splice site. The minority of the non-spliced topoisomerase IIα 3′ UTR in mouse was confirmed by Northern blot analysis. We performed transient expression assays using luciferase constructs with the mouse topoisomerase IIα 3′ genomic region, or the major spliced form of the 3′ UTR. However, neither construct affected the cell-cycle-dependent expression of the reporter gene driven by the topoisomerase IIα promoter. Our results strongly suggest that the mouse topoisomerase IIα 3′ UTR by itself is not involved in the cell-cycle-dependent mRNA stability.     

Characterization of the two H1°-encoding genes from Xenopus laevis

We have analyzed the promoter and the coding sequences of the two homologous histone H1°-encoding genes from Xenopus laevis, here termed H1°-1 and H1°-2. Both genes encode proteins of 193 amino acids and differ at just 16 amino-acid residues. Putative regulatory sequences identified in the promoter region are the same and are highly conserved. However, significant differences exist in the 5′ untranslated regions (UTR) of the transcribed sequences of these two genes, such as several deletions in the 5′-UTR of the H1°-2 gene in comparison with the H1°-1 gene 5′-UTR. The 3′-UTR is a short sequence of about 200 bp which is unexpected compared with the long 3′-UTR of mammalian H1° mRNA, but it is in the same size range as in avian H5 mRNA. Thus, the main differences between these two genes are observed in sequences potentially involved in the regulation of the H1° gene expression such as the 5′-UTR. The two genes are expressed during embryogenesis and in several adult tissues. We discuss these findings in terms of the evolution of histone H1° genes in vertebrates and the appearance of histone H5 in avian species.© 1997 Elsevier Science B.V. All rights reserved.     

Human Tudor staphylococcal nuclease (Tudor-SN) protein modulates the kinetics of AGTR1-3′UTR granule formation

Human Tudor staphylococcal nuclease (Tudor-SN) interacts with the G3BP protein and is recruited into stress granules (SGs), the main type of discrete RNA-containing cytoplasmic foci structure that is formed under stress conditions. Here, we further demonstrate that Tudor-SN binds and co-localizes with AGTR1-3′UTR (3′-untranslated region of angiotensin II receptor, type 1 mRNA) into SG. Tudor-SN plays an important role in the assembly of AGTR1-3′UTR granules. Moreover, endogenous Tudor-SN knockdown can decrease the recovery kinetics of AGTR1-3′UTR granules. Collectively, our data indicate that Tudor-SN modulates the kinetics of AGTR1-3′UTR granule formation, which provides an additional biological role of Tudor-SN in RNA metabolism during stress.     

Structure of the chicken interferon-γ gene,and comparison to mammalian homologues

The sequence of the chicken interferon-γ (ifn-γ) gene was determined, one of the first non-mammalian cytokine gene structures to be elucidated. Initial genomic clones were amplified from chicken genomic DNA and were used to isolate a cosmid clone covering the entire gene for sequencing. The exon:intron structure of chicken ifn-γ is very similar to those of its mammalian homologues, with the exception of the third intron, which is markedly shorter in the chicken. The first exon contains both 5′ UTR and signal sequence and the first 22 aa of the mature protein. The remainder of the coding region lies in exons 2–4. Exon 4 also encodes the stop codon and the 3′ UTR, including two possible polyadenylation signals. A number of potential regulatory sequences similar to those found in mammals have been identified, in the promoter, in each intron and in the 3′ UTR. In the promoter, these include the TATAATA- and CCAT-boxes, a consensus GATA motif in the reverse orientation and a potential NF-κB binding site. Other regulatory elements identified in the promoters of mammalian ifn-γ genes are absent. Internal to the gene structure, regulatory sequences identified include elements found in the DNase I hypersensitivity region of the first intron of the human ifn-γ gene and several potential NF-κB binding sites. The 3′ UTR contains an AT-rich sequence, including nine repeats of the `instability' motif ATTTA. As in mammals, chicken ifn-γ is a single copy gene. The gene is highly conserved, with no polymorphisms yet identified using either RFLP or SSCP in the coding region. However, promoter sequence polymorphisms between different inbred lines of chickens have been identified, with possible links to disease resistance.     

Effects of specific and prolonged expression of zebrafish growth factors,Fgf2 and Lif in primordial germ cells in vivo

Primordial germ cells (PGCs), specified early in development, proliferate and migrate to the developing gonad before sexual differentiation occurs in the embryo and eventually give rise to spermatogonia or oogonia. In this study, we discovered that nanos3 3′UTR, a common method used to label PGCs, not only directed PGC-specific expression of DsRed but also prolonged this expression up to 26 days post fertilization (dpf) when DsRed-nanos3 3′UTR hybrid mRNAs were introduced into 1- to 2-cell-stage embryos. As such, we employed this knowledge to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and bone morphogenetic protein 4 (Bmp4) in the PGCs and evaluate their effects on PGC development in vivo for over a period of 3 weeks. The results show that expression of Fgf2 significantly increased PGC number at 14- and 21-dpf while Bmp4 resulted in severe ventralization and death of the embryos by 3 days. Expression of Lif resulted in a significant disruption of PGC migration. Mopholino knockdown experiments indicated that Lif illicited its effect on PGC migration through Lif receptor a (Lifra) but not Lifrb. The general approach described in this study could be used to achieve prolonged PGC-specific expression of other proteins to investigate their roles in germ cell and gonad development. The results also indicate that zebrafish PGCs have a mechanism to stabilize and prolong the expression of mRNA that carries nanos3 3′UTR. Understanding this mechanism may make it possible to achieve prolonged RNA expression in other cell types.     

Identification and cytogenetic localization of vitelline membrane messenger RNAs in Drosophila

During stages 9 and 10 of oogenesis in Drosophila the major proteins involved in vitelline membrane (VM) formation are synthesized and secreted by the somatic follicle cells surrounding the oocyte. To identify potential mRNAs involved in VM protein synthesis, newly synthesized poly(A)-containing RNA from egg chambers of different developmental stages was studied. Urea-agarose gel electrophoresis revealed two RNA bands in stage 10 egg chambers in the size range expected for those which encode the smaller VM proteins. These RNA bands, T₁ and T₂, are specifically enriched in stage 10 follicle cell preparations. In vitro translations in reticulocyte lysates in the absence and presence of microsomal membranes showed both RNA bands code for products that are synthesized in precursor forms which are processed to species that comigrate with VM proteins. T₂ directed the synthesis of processed species that comigrated with the 23- to 24-kDa and 17.5-kDa VM proteins (J. Fargnoli and G. L. Waring, 1982, Dev. Biol. 92, 306–314) while the T₁ translation product comigrated with the 14-kDa protein. To determine the cytogenetic location of the genes encoding T₁ and T₂ RNAs, radiolabeled T₁ and T₂ RNAs were hybridized in situ to salivary gland chromosomes. The results suggest that the structural genes coding for the small vitelline membrane proteins are localized at two sites on the second chromosome: 39DE and 42A.     

Discovery of a new fragrance allele and development of functional markers for identifying diverse fragrant genotypes in rice

Discovery of new fragrance alleles provides important genetic resources for breeding fragrant rice. In this study, a hybrid complementation test demonstrated the association of a new fragrance allele without mutation in the coding region with flavor formation in a fragrant rice variety Nankai 138. The new allele (badh2-p-5′UTR) has a 3-bp deletion in the 5′ untranslated region and an 8-bp insertion in the promoter (?1,314 site upstream from the initiation codon). Surprisingly, we found that there is also an 8-bp insertion in the promoter of the badh2-E7 allele. We developed a new sequence tagged site functional marker to identify the badh2-p-5′UTR and badh2-E7 alleles according to the 8-bp insertion in their promoters. A cleaved amplified polymorphic sequence (AluI) functional marker targeting a common base substitution in the intron 2 of three badh2 alleles, viz. badh2-p-5′UTR, badh2-E7 and badh2-E2, was developed to identify diverse genotypes for fragrance in rice. Based on the results of sequence alignments among the three badh2 alleles, we suggest that the badh2-E7 and badh2-p-5′UTR alleles may have the same genetic origin. In addition, the genetic distance between the badh2-E7 and badh2-p-5′UTR alleles may be closer than that between the badh2-E2 and the badh2-p-5′UTR alleles, or between the badh2-E2 and the badh2-E7 alleles.     

Donkey Orchid Symptomless Virus: A Viral ‘Platypus’ from Australian Terrestrial Orchids

Complete and partial genome sequences of two isolates of an unusual new plant virus, designated Donkey orchid symptomless virus (DOSV) were identified using a high-throughput sequencing approach. The virus was identified from asymptomatic plants of Australian terrestrial orchid Diuris longifolia (Common donkey orchid) growing in a remnant forest patch near Perth, western Australia. DOSV was identified from two D. longifolia plants of 264 tested, and from at least one plant of 129 Caladenia latifolia (pink fairy orchid) plants tested. Phylogenetic analysis of the genome revealed open reading frames (ORF) encoding seven putative proteins of apparently disparate origins. A 69-kDa protein (ORF1) that overlapped the replicase shared low identity with MPs of plant tymoviruses (Tymoviridae). A 157-kDa replicase (ORF2) and 22-kDa coat protein (ORF4) shared 32% and 40% amino acid identity, respectively, with homologous proteins encoded by members of the plant virus family Alphaflexiviridae. A 44-kDa protein (ORF3) shared low identity with myosin and an autophagy protein from Squirrelpox virus. A 27-kDa protein (ORF5) shared no identity with described proteins. A 14-kDa protein (ORF6) shared limited sequence identity (26%) over a limited region of the envelope glycoprotein precursor of mammal-infecting Crimea-Congo hemorrhagic fever virus (Bunyaviridae). The putative 25-kDa movement protein (MP) (ORF7) shared limited (27%) identity with 3A-like MPs of members of the plant-infecting Tombusviridae and Virgaviridae. Transmissibility was shown when DOSV systemically infected Nicotiana benthamiana plants. Structure and organization of the domains within the putative replicase of DOSV suggests a common evolutionary origin with ‘potexvirus-like’ replicases of viruses within the Alphaflexiviridae and Tymoviridae, and the CP appears to be ancestral to CPs of allexiviruses (Alphaflexiviridae). The MP shares an evolutionary history with MPs of dianthoviruses, but the other putative proteins are distant from plant viruses. DOSV is not readily classified in current lower order virus taxa.     

Structure analysis of three T7 late mRNA ribosome binding sites

The 5′-terminal regions of the three T7 late RNA species IIIb, IV and V have been characterized. These regions contain the protein synthesis initiation sites for the T7 genes 17, 9 and 10, respectively. Each of these is located between 60 and 90 nucleotides from the 5′ terminus of an in vitro synthesized RNA species. The sequence 5′ A-C-U-U-U-A-A-G-Pu-A-G-Pu, which is common to these ribosome binding regions, contains an impressive stretch of complementarity to the sequence 5′ A-C-C-U-C-C-U-U-A, at the 3′ terminus of 16 S ribosomal RNA. The nuclease mapping technique of Wurst et al. (1978) has been used to probe intramolecular structural interactions involving these initiation regions in the RNA. My results indicate that all three initiation codons, together with other portions of the ribosome binding regions are protected, under non-denaturing conditions, against the actions of both the single-strand-specific nuclease S₁ and RNAase T₁.     

Actin-encoding cDNAs and gene expression during the intermolt cycle of the Bermuda land crab Gecarcinus lateralis

Two actin-encoding cDNAs (act1 and act2) from Gecarcinus lateralis have been sequenced or partially sequenced and the corresponding proteins deduced. The actl cDNA has a complete ORF; the act2 cDNA lacks most of the 5′ end of the coding region. The nucleotide (nt) sequences of both clones are very similar to act sequences of many organisms, the most closely related being from another arthropod, the silkmoth Bombyx mori. The proteins Actl and Act2 are more similar to vertebrate cytoplasmic actin isoforms (β-actins) than to vertebrate muscle actins (α-actins); they are also more similar to animal actins than to those of fungi or plants. Codon usage is strongly biased toward C or G in the third position. The deduced number of amino acid (aa) residues and calculated M_r for Actl are 376 aa and 41.94 kDa, respectively. The deduced aa sequence of Actl is very similar to those of muscle actins of B. mori and Drosophila melanogaster. Southern blots indicated seven to eleven act genes in the crab genome. Northern blots probed with a segment from the 3′ UTR of actl showed a single band of approx. 1.6 kb in poly(A)⁺mRNAs from epidermis, limb bud or claw muscle and in total RNAs from ovary and gill, and two bands of approx. 1.6 and 1.8 kb in total RNA from midgut gland. Western blots of one-dimensional gels of proteins from the four layers of the exoskeleton, epidermis, limb buds and claw muscle were probed with a monoclonal Ab against chicken gizzard actin; tissue- and stage-specific changes in actin content were observed. The presence of several isoforms, and differences in their number and occurrence at various stages of the intermolt cycle, were detected on Western blots of two-dimensional gels.     

Structures of genes encoding phospholipase A2 inhibitors from the serum of Trimeresurus flavoviridis snake

Inhibitors (PLIs) against snake venom gland phospholipases A₂ (PLA₂s) have been found in their sera. A cDNA encoding a PLI from Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) serum, cPLI-A, was isolated from the Tf liver cDNA library and sequenced. Northern blot analysis with cPLI-A showed that PLIs are expressed only in liver. Genes for PLIs, gPLI-A and gPLI-B, were isolated from the Tf genomic DNA library and their nucleotide (nt) sequences were determined. The genes consisted of four exons and three introns, and exon 4 encoded the carbohydrate recognition domain (CRD)-like motif. Comparison of the nt sequences between gPLI-A and gPLI-B showed that these genes are highly homologous, including introns, except that exon 3 is rich in nonsynonymous nt substitutions which are almost four times as frequent as synonymous nt substitutions. This evolutionary feature of PLI genes is different from that of venom gland PLA₂ isozyme genes in which nonsynonymous nt substitutions are spread over the entire mature protein-coding region.     

Methylomonas rapida sp. nov., a novel species of fast-growing,carotenoid-producing obligate methanotrophs with high biotechnological potential

The genus Methylomonas accommodates strictly aerobic, obligate methanotrophs, with their sole carbon and energy sources restricted to methane and methanol. These bacteria inhabit oxic-anoxic interfaces of various freshwater habitats and have attracted considerable attention as potential producers of a single-cell protein. Here, we characterize two fast-growing representatives of this genus, strains 12 and MP1^T, which are phylogenetically distinct from the currently described Methylomonas species (94.0–97.3 % 16S rRNA gene sequence similarity). Strains 12 and MP1^T were isolated from freshwater sediments collected in Moscow and Krasnodar regions, respectively. Cells of these strains are Gram-negative, red-pigmented, highly motile thick rods that contain a type I intracytoplasmic membrane system and possess a particulate methane monooxygenase (pMMO) enzyme. These bacteria grow between 8 and 45 °C (optimum 35 °C) in a relatively narrow pH range of 5.5–7.3 (optimum pH 6.6–7.2). Major carotenoids synthesized by these methanotrophs are 4,4′-diaplycopene-4,4′-dioic acid, 1,1′-dihydroxy-3,4-didehydrolycopene and 4,4′-diaplycopenoic acid. High biomass yield, of up to 3.26 g CDW/l, is obtained during continuous cultivation of MP1^T on natural gas in a bioreactor at a dilution rate of 0.22 h^?1. The complete genome sequence of strain MP1^T is 4.59 Mb in size; the DNA G + C content is 52.8 mol%. The genome encodes four rRNA operons, one pMMO operon and 4,216 proteins. The genome sequence displays 82–85 % average nucleotide identity to those of earlier described Methylomonas species. We propose to classify these bacteria as representing a novel species of the genus Methylomonas, M. rapida sp. nov., with the type strain MP1^T (=KCTC 92586^T = VKM B-3663^T).     

Complete genome sequence of a novel picorna-like virus isolated from Spodoptera exigua

The complete genome sequence and the gene organization of a novel insect picorna-like virus, Spodoptera exigua virus (SeV), were determined. The genomic RNA of the SeV was 9501 nt in length excluding the poly(A) tail and contained a single, large open reading frame (nt 392–9424) encoding a 3010 aa polyprotein. Sequence comparisons with other viral polyproteins revealed that the consensus sequences for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) proteins are found on the genome in that order from the 5′ to the 3′ end. In terms of sequence similarity, identity, and genome organization, SeV resembled insect picorna-like viruses belonging to the genus Iflavirus. A phylogenetic analysis based on the eight conserved domains in the RdRp sequence showed that SeV was most closely related to the Perina nuda virus and Ectropis obliqua picorna-like virus, suggesting that these three insect picorna-like viruses might share a common ancestor.