The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.     

Specificity of recognition sequence forEscherichia coli primase

Summary We have surveyed the frequency of each of 64 trinucleotide permutations at every nucleotide frame located from 1 to 15 nucleotides upstream of primer RNA-DNA transition sites mapped within a 1.5 kb region of the bacteriophage lambda genome and a 1.4 kb region of theEscherichia coli genome. We have demonstrated that in both systems initiation of DNA synthesis strongly correlates with a CAG sequence located 11 nucleotides upstream of the DNA start sites. Based on the examination of various reports of the priming reaction catalyzed byE. coli primase in vivo and in vitro, we propose that (i)E. coli primase itself recognizes a 3′GTC 5′ sequence on the template strand, (ii) DnaB helicase releases the specificity ofE. coli primase and, (iii) the consensus recognition sequence forE. coli primase associated with DnaB helicase is 3′PuPyPy 5′.     

Sequence characteristics and divergent evolution of the chloroplastpsbA-trnH noncoding region in gymnosperms

ThepsbA-trnH intergenic region is among the most variable regions in the gymnosperm chloroplast genome. It is proposed as suitable for DNA barcoding studies and is useful in phylogenetics at the species level. This region consists of two parts differing in their evolutionary characteristics: 1) thepsbA 3′UTR (untranslated region) and 2) thepsbA-trnH intergenic spacer. We compared the sequence and RNA secondary structure of thepsbA 3′ UTR across gymnosperms and found consensus motifs corresponding to the stem portions of the RNA stem-loop structures and a consensus TGGATTGTTATGT box. ThepsbA-trnH spacer is highly variable in length and composition. Tandem repeats that form stem—loop structures were detected in both thepsbA 3′ UTR and the psbA-trnH spacer. The presence of promoters and stem—loop structures in the psbA-trnH spacer and high sequence variation in this region suggest that psbA and trnH in some gymnosperms are independently transcribed. Acomparison of chloroplast UTRs across gymnosperms offer clues to the identity of putative regulatory elements and information on selective constraints imposed on the chloroplast non-coding regions. The present study should inspire researchers to explore the full potential of thepsbA-trnH non-coding sequence and to further stimulate its application in a broader spectrum of studies, not limited to phylogenetics and DNA barcoding.     

Strong Expression and Conserved Regulation of <Emphasis Type="Italic">ACT2</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Arabidopsis ACT2 represents an ancient class of vegetative plant actins and is strongly and constitutively expressed in almost all Arabidopsis sporophyte vegetative tissues. Using the beta glucuronidase report system, the studies showed that ACT2 5′ regulatory region was significantly more active than CaMV 35S promoter in Arabidopsis seedlings and gametophyte vegetative tissues of Physcomitrella patens. Its activity was also observed in rice and maize seedlings. Thus, the ACT2 5′ regulatory region could potentially serve as a strong regulator to express a transgene in divergent plant species. ACT2 5′ regulatory region contained 15 conserved sequence elements, an ancient intron in its 5′ un-translated region (5′ UTR), and a purine-rich stretch followed by a pyrimidine-rich stretch (PuPy). Mutagenesis and deletion analysis illustrated that some of the conserved sequence elements and the region containing PuPy sequences played regulatory roles in Arabidopsis. Interestingly, mutation of the conserved elements did not lead a dramatic change in the activity of ACT2 5′ regulatory region. The ancient intron in ACT2 5′ UTR was required for its strong expression in both Arabidopsis and P. patens, but did not fully function as a canonical intron. Thus, it was likely that some of the conserved sequence elements and gene structures had been preserved in ACT2 5′ regulatory region over the course of land plant evolution partly due to their functional importance. The studies provided additional evidences that identification of evolutionarily conserved features in non-coding region might be used as an efficient strategy to predict gene regulatory elements.     

Involvement of two differenturf-s related mitochondrial sequences in the molecular evolution of the CMS-specificS-Pcf locus in petunia

In petunia, a mitochondrial (mt) locus,S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). TheS-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF,Pcf, contains parts of theatp9 andcoxII genes and an unidentified reading frame,urf-s. The second and third ORFs contain NADH dehydrogenase subunit 3 (nad3) and ribosomal protein S12 (rps12) sequences, respectively. Thenad3 andrps12 sequences included in theS-Pcf locus are identical to the corresponding sequences on the mt genome of fertile petunia. In both CMS and fertile petunia, only a single copy ofnad3 andrps12 has been detected on the physical map of the main mt genome. The origin of theurf-s sequence and the molecular events leading to the formation of the chimericS-Pcf locus are not known. This paper presents evidence indicating that two different mt sequences, related tourf-s and found in fertile petunia lines (orf-h and Rf-1), might have been involved in the molecular evolution of theS-Pcf locus. Southern analysis of mtDNA derived from both fertile and sterile petunia plants suggests that one of theseurf-s related sequences (showing 100% homology tourf-s and termedorf-h) is located on a sublimon. An additional, low-homologyurf-s related sequence (Rf-1) is shown to be located on the main mt genome 5′ to thenad3 gene. It is, thus, suggested that the sequence of events leading to the generation of theS-Pcf locus might have involved introduction of theorf-h sequence, via homologous recombination, into the main mt genome 5′ tonad3 at the region where the Rf-1 sequence is located. Contribution [No. 1581-E (1995 series)] from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel 50 250     

Emergence of a mutagenic ochre suppressor mutation under lactose selection in appm mutant ofEscherichia coli harbouring the F′lacZU118 episome

WhenEscherichia coli harbouring theppm (earlier calledadi) mutation and the F′lacZU118 episome is subjected to lactose selection in the presence of suboptimal concentrations of glycerol, Lac⁺ colonies emerge after 5–6 days. They are shown to harbour an ochre suppressor mutation at 15.15 min. Inactivation ofrecA results in approximately four-fold reduction in the response. In theppm — ochre suppressor double mutant background the leakiness of thelacZ allele carried by F′ CC105 is enhanced, suggesting misreading of a valine codon (GUG) as glutamic acid codon (GAG). This is accompanied by reversion of thelacZ mutation tolacZ ⁺ (GTG → GAG). In LB medium both the leakiness and reversion are inhibited by streptomycin. Inactivation ofrecA did not affect leakiness but abolished reversion. These data are discussed in relation to the importance of allele leakiness and restricted growth in stationary-phase (adaptive) mutagenesis.     

A full lengthTy3/gypsy-type retrotransposon in the fernAdiantum

Ty3/gypsy-type LTR-retrotransposons have been found only in lily and maize but not in cryptogam. In fernAdiantum, we recently found a full-lengthTy3/gypsy-type LTR-retrotransposon (ARET-1; 8284 bp). This retrotransposon has both 5′ and 3′ LTRs (1.2 kb), a primer binding site, a polypurine tract, and an RNA binding motif and its domain arrangement in thepol region is the same as that ofTy3/gypsy-type retrotransposon. These results suggest thatTy3/gypsy-type retrotransposons are widespread among vascular plants. The nucleotide sequence data reported will appear in the EMBL, DDBJ and GenBank Nucleotide Sequence Databases under the accession number AB003364.     

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.     

Cloning and expression of the Apa LI, Nsp I, Nsp HI, Sac I, Sca I, and Sap I restriction-modification systems in Escherichia coli

The genes encoding the ApaLI (5′-G^TGCAC-3′), NspI (5′-RCATG^Y-3′), NspHI (5′-RCATG^Y-3′), SacI (5′-GAGCT^C-3′), SapI (5′-GCTCTTCN1^-3′, 5′-^N4GAAGAGC-3′) and ScaI (5′-AGT^ACT-3′) restriction-modification systems have been cloned in E.␣coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5′-CATG-3′) restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5′-GCTCTTC-3′ blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene. Received: 15 April 1998 / Accepted: 3 August 1998     

Molecular analysis of theBg-rbg transposable element system ofZea mays L.

Summary The two components of theBg-rbg transposable element system of maize have been cloned. TheBg element, isolated from the mutable allelewx-m32 :: Bg is inserted in the intron of theWaxy (Wx) gene between exons 12 and 13. The length of the element is of 4869 bp.Bg has 5 by terminal inverted repeats, and generates upon insertion an 8 by direct duplication of the target sequence. Both ends of theBg element contain a 76 by direct repeat adjacent to the terminal inverted repeats. The hexamer motif TATCG_kC ^G is here repeated several times in direct or inverse orientation. Therbg element was isolated from the mutable alleleo2m(r) where it is located in the promoter region of theOpaque-2 (O2) gene.rbg is approximately 4.5 kb in length, has terminal inverted repeats identical to those of theBg element, and is also flanked by an 8 by direct duplication at the target site. LikeBg, rbg carries the 76 by direct repeats. Restriction enzyme analysis reveals that, compared toBg, the receptor element is distinguishable by small deletion and insertion events. Sequence data indicate that not more than 75% homology exists at the DNA level between therbg element and the autonomousBg element.     

Isolation and characterisation of an aryl-β-D-glucoside uptake and utilisation system ( abg ) from the gram-positive ruminal Clostridium species C. longisporum

A phosphotransferase-dependent aryl-β-glucoside uptake and utilisation system (abg) was isolated from the ruminal Clostridium (“C. longisporum”). The system is composed of three genes, abgG, abgF and abgA, and a number of regulatory regions, including terminator/antiterminator type stem-loop structures preceding the abgG and abgF genes. Similarity analysis of the proteins encoded by these genes indicated that they were responsible for the regulation of the abg system through antitermination (AbgG), the uptake and phosphorylation of aryl-β-glucosides (AbgF) and the hydrolysis of the intracellular phosphorylated glycosides (AbgA). Experimental evidence for the functions of AbgF and AbgA was obtained. Although it was not possible to demonstrate any function for AbgG, a promoter 5′ to the abgG gene was identified which was responsible for expression of the downstream genes. The abg system is remarkably similar to operons from the gram negative Enterobacteriaceae, both in the coding and non-coding regulatory regions. Received: 3 April 1997 / Accepted: 8 September 1997     

The architecture of the chloroplast psbA-trnH non-coding region in angiosperms

The psbA-trnH intergenic region is among the most variable regions in the angiosperm chloroplast genome. It is a popular tool for plant population genetics and species level phylogenetics and has been proposed as suitable for DNA barcoding studies. This region contains two parts differing in their evolutionary conservation: 1) the psbA 3′UTR (untranslated region) and 2) the psbA-trnH intergenic non-transcribed spacer. We compared the sequence and RNA secondary structure of the psbA 3′ UTR across angiosperms and found consensus motifs corresponding to the stem portions of the RNA stem-loop structures and a consensus TTAGTGTATA box. The psbA-trnH spacer exhibited patterns that can be explained by the independent evolution of large inversions in the psbA 3′UTR and mutational hot spots in the remaining portion of the psbA-trnH spacer. We conclude that a comparison of chloroplast UTRs across angiosperms offer clues to the identity of putative regulatory elements and information about selective constraints imposed on the chloroplast non-coding regions.     

Analysis of transitive RNA silencing after grafting in transgenic plants with the coat protein gene of <Emphasis Type="Italic">Sweet potato feathery mottle virus</Emphasis>

We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5′ 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5′ or 3′ silencing inducer rootstocks. When non-silenced scions were grafted onto 5′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3′ region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3′ region of the transgene and did not spread to the 5′ region. In addition, results from crossing experiments, involving non-silenced and 3′ silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5′–3′ direction, not in the 3′–5′ direction, along the transgene mRNA.     

Development of a simple molecular marker specific for detecting the self-compatible<Emphasis Type="Italic">S4′</Emphasis> haplotype in sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

In sweet cherry (Prunus avium L.), theS4′ haplotype, characterized by a self-incompatibility (SI) defect in pollen, is self-compatible and is derived from the self-incompatibleS4 haplotype by x-ray mutagenesis.SFBs (S haplotype-specific F-box protein genes) have been found to associate with pollen determinant of SI. This report identified theSFB4′ of the self-compatibleS4′ haplotype. The alignment of the sequences ofSFB4′ andSFB4 by the BLAST program revealed a 4-bp deletion inSFB4′, which is TTTA. The sequence polymorphism generated by the TTTA deletion inSFB4′ was exploited to develop a simple molecular marker specific for detecting theS4′ but not theS4 haplotype. The simple marker specific to theS4′ haplotype can be visualized directly on an agarose gel, so it can be immediately applied to a marker-assistant cherry-breeding program. Thus, this work provides a practical molecular marker for cherry breeding. Principal author. An erratum to this article is available at .     

Comparative analysis in cereals of a key proline catabolism gene.

Proline accumulation and catabolism play significant roles in adaptation to a variety of plant stresses including osmotic stress, drought, temperature, freezing, UV irradiation, heavy metals and pathogen infection. In this study, the gene Δ¹ -pyrroline-5-carboxylate dehydrogenase (P5CDH), which catalyzes the second step in the conversion of proline to glutamate, is characterized in a number of cereal species. P5CDH genes from hexaploid wheat, Triticum turgidum (durum wheat), Aegilops tauschii, Triticum monococcum, barley, maize and rice were shown to be conserved in terms of gene structure and sequence, present as a single copy per haploid, non-polyploid genome and located in evolutionarily conserved linkage groups. A wheat cDNA sequence was shown by yeast complementation to encode a functional P5CDH activity. A divergently-transcribed rab7 gene was identified immediately 5′ of P5CDH in all grasses examined, except rice. The rab7/P5CDH intergenic region in these species, which presumably encompasses 5′ regulatory elements of both genes, showed a distinct pattern of sequence evolution with sequences in juxtaposition to each ORF conserved between barley, wheat, A. tauschii and T. monococcum. More distal 5′ sequence in this intergenic region showed a higher rate of divergence, with no homology observed between these regions in the wheat and barley genomes. Maize and rice showed no similarity in regions 5′ of P5CDH when compared with wheat, barley, and each other, apart from a 22 bp region of conserved non-coding sequence (CNS) that is similar to a proline response element identified in the promoter of the Arabidopsis proline dehydrogenase gene. A palindromic motif similar to this cereal CNS was also identified 5′ of the Arabidopsis AtP5CDH gene showing conservation of this sequence in monocot and dicot lineages.     

A new mutation inEscherichia coli K12,isfA, which is responsible for inhibition of SOS functions

A new mutation inEscherichia coli K12,isfA, is described, which causes inhibition of SOS functions. The mutation, discovered in a ΔpolA ⁺ mutant, is responsible for inhibition of several phenomena related to the SOS response inpolA ⁺ strains: UV- and methyl methanesulfonate-induced mutagenesis, resumption of DNA replication in UV-irradiated cells, cell filamentation, prophage induction and increase in UV sensitivity. TheisfA mutation also significantly reduces UV-induced expression of β-galactosidase fromrecA::lacZ andumuC′::lacZ fusions. The results suggest that theisfA gene product may affect RecA^* coprotease activity and may be involved in the regulation of the termination of the SOS response after completion of DNA repair. TheisfA mutation was localized at 85 min on theE. coli chromosome, and preliminary experiments suggest that it may be dominant to the wild-type allele.