Downstream Copy Research Articles

Interspecies transfer of RAMOSA1 orthologs and promoter cis sequences impacts maize inflorescence architecture.

Grass inflorescences support floral structures that each bear a single grain, where variation in branch architecture directly impacts yield. The maize (Zea mays) RAMOSA1 (ZmRA1) transcription factor acts as a key regulator of inflorescence development by imposing branch meristem determinacy. Here, we show RA1 transcripts accumulate in boundary domains adjacent to spikelet meristems in sorghum (Sorghum bicolor, Sb) and green millet (Setaria viridis, Sv) inflorescences similar as in the developing maize tassel and ear. To evaluate the functional conservation of syntenic RA1 orthologs and promoter cis sequences in maize, sorghum, and setaria, we utilized interspecies gene transfer and assayed genetic complementation in a common inbred background by quantifying recovery of normal branching in highly ramified ra1-R mutants. A ZmRA1 transgene that includes endogenous upstream and downstream flanking sequences recovered normal tassel and ear branching in ra1-R. Interspecies expression of two transgene variants of the SbRA1 locus, modeled as the entire endogenous tandem duplication or just the nonframeshifted downstream copy, complemented ra1-R branching defects and induced unusual fasciation and branch patterns. The SvRA1 locus lacks conserved, upstream noncoding cis sequences found in maize and sorghum; interspecies expression of a SvRA1 transgene did not or only partially recovered normal inflorescence forms. Driving expression of the SvRA1 coding region by the ZmRA1 upstream region, however, recovered normal inflorescence morphology in ra1-R. These data leveraging interspecies gene transfer suggest that cis-encoded temporal regulation of RA1 expression is a key factor in modulating branch meristem determinacy that ultimately impacts grass inflorescence architecture.

Intron gain by tandem genomic duplication: a novel case in a potato gene encoding RNA-dependent RNA polymerase.

The origin and subsequent accumulation of spliceosomal introns are prominent events in the evolution of eukaryotic gene structure. However, the mechanisms underlying intron gain remain unclear because there are few proven cases of recently gained introns. In an RNA-dependent RNA polymerase (RdRp) gene, we found that a tandem duplication occurred after the divergence of potato and its wild relatives among other Solanum plants. The duplicated sequence crosses the intron-exon boundary of the first intron and the second exon. A new intron was detected at this duplicated region, and it includes a small previously exonic segment of the upstream copy of the duplicated sequence and the intronic segment of the downstream copy of the duplicated sequence. The donor site of this new intron was directly obtained from the small previously exonic segment. Most of the splicing signals were inherited directly from the parental intron/exon structure, including a putative branch site, the polypyrimidine tract, the 3′ splicing site, two putative exonic splicing enhancers, and the GC contents differed between the intron and exon. In the widely cited model of intron gain by tandem genomic duplication, the duplication of an AGGT-containing exonic segment provides the GT and AG splicing sites for the new intron. Our results illustrate that the tandem duplication model of intron gain should be diverse in terms of obtaining the proper splicing signals.

Homologous Recombination Assay

Repair of double strand break by homologous recombination was examined using U2OS cells or RG37 cells harbouring specific substrate developed by Puget et al. (2005) and Dumay et al. (2006), respectively, to measure the repair of DNA double strand breaks by homologous recombination. The substrate is composed of two inactive copies of the GFP gene. The upstream copy is inactive due to the absence of promoter, the downstream copy present a promoter but is inactivated by the insertion of the sequence coding for the recognition site of the I-SceI enzyme. The substrate is stably expressed in cells after its insertion in the genome and present as a unique copy. The unique DNA double strand break is then induced by the expression of the I-SceI enzyme after cell transfection with a plasmid coding for the I-SceI enzyme., 使用由Puget等人开发的含有特异性底物的U2OS细胞或RG37细胞检查通过同源重组修复双链断裂。 （2005）和Dumay等人 （2006），分别通过同源重组来测量DNA双链断裂的修复。 底物由GFP基因的两个无活性拷贝组成。 上游拷贝由于不存在启动子而不活跃，下游拷贝存在启动子，但通过插入编码I-SceI酶的识别位点的序列而失活。 底物在插入基因组后稳定地表达在细胞中，作为唯一拷贝存在。 然后通过用编码I-SceI酶的质粒转染细胞后，通过I-SceI酶的表达来诱导独特的DNA双链断裂。

Genes Involved in the Degradation of Ether Fuels by Bacteria of the Mycobacterium/Rhodococcus Group

A cluster of genes encoding a cytochrome P-450 monooxygenase system involved in the utilisation of ethyl tert-butyl ether (ETBE) was cloned in Rhodococcus ruber IFP 2001. This cluster includes ethR, a putative regulator gene of the araC/xylS family; ethA, encoding a ferredoxin reductase; ethB, encoding a cytochrome P-450, ethC, encoding a ferredoxin; and ethD, which is required for the function of the monooxygenase system, but whose exact role is unknown. The ethRABCD cluster is flanked on either side by two identical copies of a class II transposon, which explains that it is lost at high frequency by homologous recombination when the strain is grown under non selective conditions. Two other, highly conserved clusters of eth genes were detected in the ETBE-utilizing strains Rhodococcus zopfii IFP 2005 and Mycobacterium sp. IFP 2009. In all cases, the eth locus is inserted in a different genomic context, suggesting that it may be transferred horizontally between different species and inserted at different sites in the genome. In addition, in R. zopfii IFP 2005, the downstream copy of the transposon carries a 117-bp (base pairs) deletion; in Mycobacterium sp. IFP 2009, the upstream copy is absent and the downstream copy is inserted 2771 bp closer to the ethRABCD cluster.

A partial HPRT gene duplication generated by non-homologous recombination in V79 chinese hamster cells is eliminated by homologous recombination

Here, the sequence in the hprt gene of the duplication mutant SPD8 originating from V79 Chinese hamster cells was determined. The duplication arose after non-homologous recombination between exon 6 and intron 7, resulting in an extra copy of the 3′ portion of exon 6, of exon 7 and of flanking intron regions. Only a duplication of exon 7 is present in the mRNA, since the duplicated exon 6 lacks its 5′ splice site and is removed during RNA processing. The findings in this study suggest that the non-homologous recombination mechanism which occurred here may have been initiated by endonucleases, rather than by a spontaneous double strand break. Subsequently, 14 spontaneous SPD8 revertants with a functional hprt gene were isolated and characterized using PCR and sequencing. The data revealed that although the SPD8 cell line arose by non-homologous recombination, it reverts spontaneously by homologous recombination. Interestingly, the downstream copy of exon 7 was restored by this process. This was indicated by the presence of a specific mutation, a T-to-G transversion, close to the breakpoint, a characteristic unique to the SPD8 clone. Our results suggest that the spontaneous reversion of this cell line by homologous recombination may involve an exchange, rather than a conversion mechanism.

Long-distance effect of downstream transcription on activity of the supercoiling-sensitive leu-500 promoter in a topA mutant of Salmonella typhimurium

Expression of the lacZ gene from the supercoiling-sensitive leu-500 promoter on a plasmid in topA mutant cells was stimulated by activating a divergently oriented Tac promoter, 400 bp upstream from leu-500. The stimulation was approximately threefold regardless of whether the Tac promoter drove the expression of the tet gene, whose product is membrane bound, or of the cat gene, whose product is cytosolic. Putting a second copy of the Tac promoter downstream from lacZ, approximately 3,000 bp from leu-500 in the same orientation as the latter, resulted in 30-fold increase in lacZ expression upon isopropyl-beta-D-thiogalactopyranoside induction. Again, these effects were independent of the nature of the gene upstream from leu-500 (tet or cat). With both tet- and cat-harboring constructs, activation of the two Tac promoter copies caused plasmid DNA to become hypernegatively supercoiled in topA mutant cells. Thus, neither leu-500 activation nor hypernegative plasmid DNA supercoiling appears to require membrane anchoring of DNA in this system. Replacing the downstream copy of Tac with a constitutive promoter resulted in high-level lacZ expression even when the upstream copy was repressed. Under these conditions, no hypernegative DNA supercoiling was observed, indicating that the activity of plasmid-borne leu-500 in topA mutant cells does not necessarily correlate with the linking deficit of plasmid DNA. The response of the leu-500-lacZ fusion to downstream transcription provides a sensitive assay for transcriptional supercoiling in bacteria.

Hepadnavirus reverse transcription initiates within the stem-loop of the RNA packaging signal and employs a novel strand transfer.

Replication of the hepadnavirus genome occurs by reverse transcription of an RNA pregenome and is mediated by the viral polymerase; the polymerase is also required for packaging of the pregenome through interaction with the RNA packaging signal, epsilon. Previous work suggested that reverse transcription of minus-strand DNA initiates within the sequence element DR1 (direct repeat 1) and that disruption of DR1 activates a cryptic initiation site in a downstream copy of epsilon. However, using active duck hepatitis B virus polymerase expressed in a yeast Ty vector system, we demonstrate that synthesis of minus-strand DNAs with 5' ends at DR1 requires the stem-loop of epsilon, whereas the production of DNAs mapping to epsilon does not require DR1. Mutations at epsilon that remove homology between epsilon and DR1 eliminate reverse transcripts with 5' ends in DR1, and restoring homology at DR1 to a mutant epsilon partially restores DNAs mapping to DR1. Insertions of one nucleotide into the bulge region of the epsilon stem-loop increase the length of minus-strand DNA whose 5' ends map to DR1 by one nucleotide. Thus, very short minus-strand primers are initiated within epsilon, rather than in DR1 as previously supposed; they are then transferred to a four-nucleotide homology in DR1. Transfer was also observed in vivo during replication of duck hepatitis B virus in avian cells; in this case, transfer is from the 5' copy of epsilon to the 3' copy of DR1. This minus-strand transfer reaction is likely to be a general feature of all hepadnaviruses.

Repetitive sequence involvement in the duplication and divergence of mouse lysozyme genes.

Mouse M and P lysozymes are the products of separate genes, are specifically expressed in separate tissues, and are adapted to different functions. The lysozyme genes have assumed these markedly different characteristics following their generation by gene duplication 30-50 million years ago. The discovery of the lysozyme P gene only 5 kb upstream from the M gene in tandem repeat has enabled an investigation of the molecular basis of their duplication and subsequent divergence. The duplication is shown to have involved recombination between two B2 repeat sequences flanking the original gene. The resulting downstream copy has retained the myeloid specificity of expression along with just 1.7 kb of upstream sequences, while the upstream copy is inactive in macrophages and has become expressed instead in the small intestine. Although multiple gene conversion events have served to maintain a generally high homology between the genes, certain regions have been found to be specific for either one of the gene pair: two repetitive sequences peculiar to the P region may serve to protect the coding regions from gene conversion, while sequences unique to the M gene may be more directly involved in differential regulation.

Detailed structural analysis of a genome rearrangement in bovine rotavirus

A genome rearrangement involving RNA segment 11 of a bovine rotavirus has been analysed by molecular cloning and sequencing. This revealed that the rearranged genome segment was generated by a head to tail concatemerisation of two almost full length copies of segment 11. The upstream copy of the gene has lost its 3' end and the downstream copy its 5' end. The truncation of the upstream copy of the gene occurs within the termination codon for VP11 converting it from a UAG to a UGA, the rearranged gene is therefore still able to encode a normal VP11. The possible mechanisms by which this rearrangement may have been generated are discussed.

Novel rearrangements of IS30 carrying plasmids leading to the reactivation of gene expression.

Unusual DNA rearrangements involving the prokaryote mobile genetic element IS30 have been identified. In order to study the potential mechanisms for the reactivation of a gene after IS element insertion, IS30 was introduced between the lacUV5 promoter and the galK gene of the multicopy plasmid pFR100. In this plasmid terminators of RNA transcription in the sequence of IS30 prevent expression of the galK gene from the lacUV5 promoter. A number of independently isolated mutant plasmids re-expressing the galK gene were studied and shown to be tandemly repeated dimers of the original plasmid. However, the two copies of IS30 were tandemly repeated at one of the original sites of insertion, while at the other site IS30 and three base pairs were missing. The repeated copies of IS30 were separated by two base pairs, the same as those originally flanking the elements. An apparently identical mechanism generated cointegrates between a derivative of plasmid pFD51 carrying IS30 upstream of a promoterless galK gene and a derivative of plasmid pACYC177 carrying IS30 inserted into the beta-lactamase gene. This arrangement brought the galK gene under the control of the beta-lactamase promoter of pACYC177. A mechanism involving aborted conservative transposition of IS30 is discussed as a possible route for the generation of these novel cointegrates. In a third experiment we isolated an insert of IS30 which was also two base pairs away from an already resident IS30 element. This insertion of IS30 created a strong promoter of RNA transcription, which has the potential to increase the expression of the transposase in the downstream copy of the element.

DNA environment of the aerobactin iron uptake system genes in prototypic ColV plasmids.

The aerobactin iron uptake system genes in the prototypic plasmid pColV-K30 are flanked by inverted copies of insertion sequence IS1 and by two distinct replication regions. To address the question of how these flanking regions may facilitate the maintenance and spread of the aerobactin system among the plasmids and chromosomes of enteric species, we investigated the DNA environment of 12 ColV plasmids. We found that the aerobactin system-specific genes are conserved in every plasmid phenotypically positive for the aerobactin system. The upstream IS1 and its overlapping replication region (REPI) are also conserved. This replication region was cloned from several ColV plasmids and found to be functional by transforming these cloned derivatives into a polA bacterial host. In contrast, the downstream flanking region is variable. This includes the downstream copy of IS1 and the downstream replication region (REPII). We infer from these results that sequences in addition to the two flanking copies of IS1, in particular the upstream region including REPI, have been instrumental in the preservation and possible spread of aerobactin genes among ColV plasmids and other members of the FI incompatibility group.

Maturation of Escherichia coli tryptophan operon mRNA: evidence for 3' exonucleolytic processing after rho-dependent termination.

The mature 3' end of Escherichia coli tryptophan operon mRNA in vivo coincides with a site (trp t) having features commonly associated with rho-independent terminators in bacteria. Efficient generation of this 3' end in vivo is nevertheless affected by a distal rho-dependent site (trP-t'), though these two sites behave independently in vitro. We have cloned these sites upstream of the galactokinase gene (galK), and galactokinase levels in vivo indicate that, as terminators per se, their efficiencies (37% for trp t, and 79% for trp t') do not differ significantly from those observed in vitro. However, when the trp t hairpin is placed between galK and a downstream copy of trp t', galactokinase levels are enhanced 2- to 3-fold. This suggests the involvement of a post-transcriptional event, such as RNA processing, in determining the level of gene activity. Indeed, in the presence of the 3' exonuclease RNase II, mRNA terminated by rho factor in vitro at the trp t' site is processed back to the trp t site. The remote trp t' region appears to be the major termination site for trp mRNA, and the trp t hairpin serves a dual function-as a minor terminator, and as a protective barrier to 3' exonucleolytic degradation. We infer that the tandem terminators, rho factor, and RNA processing are all required to generate the mature 3' end of this bacterial mRNA.

Cis-Acting RNA packaging locus in the 115-nucleotide direct repeat of Rous sarcoma virus.

The v-src gene of Schmidt-Ruppin strain A of Rous sarcoma virus is flanked by a 115-nucleotide direct repeat. Mutants that lack either the upstream or downstream copy replicate normally. However, mutants that lack both copies do not replicate. Cloned viral DNA lacking both copies of the 115-nucleotide sequence is capable of directing the transcription of viral RNA posttransfection. This viral RNA is polyadenylated, spliced, exported from the nucleus, and translated into protein normally. However, virions isolated from the culture medium 48 h posttransfection lack viral RNA. When mutant DNA is contransfected with wild-type DNA, the virions produced 48 h later contain wild-type RNA but not mutant RNA, even though both RNAs are present in the cytoplasm. We propose that the 115-nucleotide element of Rous sarcoma-avian leukosis virus encodes a cis-acting sequence that is necessary for the proper incorporation of viral RNA into virions.

Control of bacteriophage γ repressor establishment transcription

The kinetics of lambda l-strand repressor establishment RNA synthesis were measured from the y-cII region of induced tof- prophage. The activity of the repressor is epistatic to the expression of gene tof coding for the antirepressor (Tof). The activity of Tof, is epistatic to the expression of repressor gene cI transcription from Prm and the expression of repressor establishment transcription from a site 600 to 800 nucleotides upstream from Prm. Three modes of l-strand rex-cI-tof-y-cII-oop transcription occur: (a) Prm promoted cI-rex mRNA synthesis from noninduced prophage, (b) coordinate lit and oop synthesis from induced tof+ prophage and (c) establishment transcription from induced tof- prophage. The synthesis or stability of oop RNA is much reduced from induced tof-, compared with tof+ prophage. The oop transcription from tof- prophage is not coordinate with RNA synthesis from the y-cII interval. The y-cII-(oop) portion of the establishment transcript appears more unstable than the translated downstream copy of genes rex-cI. The initiation of any repressor establishment transcription requires the products of lambda genes cIII, cII, P and Escherichia coli genes dnaB, dnaG, but not actual lambda DNA synthesis. This result demonstrates that common factors, i.e. replication gene products, are required for the initiation of establishment transcription, lambda replication and lit, oop RNA synthesis; and explains why cIII+ cI+ cII+ replication defective phage lysogenize poorly at low multiplicities of infection. The cIII and cII products were shown to act after an earlier replication initiation or activation event. Repressor establishment transcription and repressor mRNA synthesis from Prm (from induced cI- tof-, cIII- cI- tof- or cI- tof- cii- prophage) are amplified by gene dosage. The extent of lysogenization of E. coli by lambda cIII-, cII- or replication minus mutants, defective for initiation of establishment synthesis, is attributed to gene dosage dependent transcription from Prm. The mechanism by which Tof inhibits the initiation of establishment transcription does not appear to require repression of RNA synthesis from PL and PR. RNA synthesis from these promoters is blocked by renaturation of the repressor 5 min after induction, before establishment transcription is detected; however, establishment RNA synthesis measured between 12-13 min after induction, i.e. 7 min after renaturation of the repressor, is only partially reduced.