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At least 145 records · Page 8Linked to original sources
PubMed1989

Characterization of the genetic organization of the HindIII M region of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata reveals major differences among baculoviruses.

A region including the 4 kb HindIII M fragment of the multicapsid nuclear polyhedrosis virus (MNPV) of Orgyia pseudotsugata (OpMNPV) genome was sequenced, transcriptionally mapped, and compared to the homologous region in the MNPV of Autographa californica (AcMNPV). Five open reading frames (ORFs) oriented in the same direction were identified and were found to be expressed at late times post-infection. The mRNAs from the five ORFs were found to coterminate at a single site downstream of ORF 5. The conserved late gene promoter/mRNA start site sequence (AGTAAG) was present upstream of all the ORFs, but did not appear to be the major site of mRNA initiation for two of the ORFs as determined by primer extension analysis. These data indicated that use of this signal for transcriptional initiation may vary between different ORFs. The predicted amino acid sequences for the five ORFs of AcMNPV and OpMNPV were compared and amino acid homologies of 26 to 72% were observed. The comparison revealed a number of major differences in the genomes of the two viruses. A putative transposable element of 634 nucleotides was found to be inserted into the previously reported AcMNPV ORF 1 sequence. In addition, it was found that a region corresponding to the 4 kb HindIII K/EcoRI S/hr5 region of AcMNPV was not present in the OpMNPV genome.

Amino Acid Sequence↗
PubMed1997

A highly conserved DNA replication module from Streptococcus thermophilus phages is similar in sequence and topology to a module from Lactococcus lactis phages.

A highly conserved DNA region extending over 5 kb was observed in Streptococcus thermophilus bacteriophages. Comparative sequencing of one temperate and 26 virulent phages demonstrated in the most extreme case an 18% aa difference for a predicted protein, while the majority of the phages showed fewer, if any aa changes. The relative degree of aa conservation was not homogeneous over the DNA segment investigated. Sequence analysis of the conserved segment revealed genes possibly involved in DNA transactions. Three predicted proteins (orf 233, 443, and 382 gene product (gp)) showed nucleoside triphosphate binding motifs. Orf 443 gp showed in addition a DEAH box motif, characteristically found in a subgroup of helicases, and a variant zinc finger motif known from a phage T7 helicase/primase. Tree analysis classified orf 443 gp as a distant member of the helicase superfamily. Orf 382 gp showed similarity to putative plasmid DNA primases. Downstream of orf 382 a noncoding repeat region was identified that showed similarity to a putative minus origin from a cryptic S. thermophilus plasmid. Four predicted proteins showed not only high degrees of aa identity (34 to 63%) with proteins from Lactococcus lactis phages, but their genes showed a similar topological organization. We interpret this as evidence for a horizontal gene transfer event between phages of the two bacterial genera in the distant past.

Amino Acid Sequence↗
PubMed1996

Alloplasmic male-sterile Brassica lines containing B. tournefortii mitochondria express an ORF 3' of the atp6 gene and a 32 kDa protein. off.

Analyses of mitochondrial transcription and in organello translation were performed with the Brassica tournefortii cytoplasm. This cytoplasm causes alloplasmic male sterility when combined with the nuclear genomes of B. napus and B. juncea. Mitochondrial RNA and protein banding patterns were compared between the fertile wild species B. tournefortii, an alloplasmic male-sterile B. juncea line, an alloplasmic male-sterile B. napus line and an alloplasmic B. napus line with restored fertility. The analyses were carried out to identify differences in gene expression and to investigate whether alterations in gene expression accompanied male sterility. A difference in transcription patterns between the fertile B. tournefortii and the alloplasmic lines was found for the atp6 gene. The atp6 region was investigated further, since a similar alteration in atp6 transcription has been observed in two other Brassica cytoplasms which are associated with cytoplasmic male sterility (CMS). The additional longer atp6 transcript detected in the alloplasmic lines in the present study was found to contain an open reading frame (ORF) located downstream of the atp6 gene. DNA sequencing revealed that the ORF, orf263, could encode a protein with a predicted molecular weight of about 29 kDa. In organello analysis detected two proteins of 29 and 32 kDa respectively, which were found only in the alloplasmic lines. Furthermore, the 32 kDa protein accompanied male sterility since it was absent in alloplasmic plants restored to fertility. The protein analysis might indicate that orf263 is translated and causes CMS.

Amino Acid Sequence↗
PubMed2004

PilR enhances the sensitivity of Xanthomonas axonopodis pv. citri to the infection of filamentous bacteriophage Cf.

The pilA gene, which encodes the major structure of pili, is required for infection of Xanthomonas axonopodis pv. citri ( X. a. pv. citri) by the filamentous bacteriophage Cf. Two open reading frames (ORFs) located downstream of pilA were cloned and characterized. One 1392-bp ORF encodes a protein of 464 amino acids which shares substantial similarity with pilR of other bacterial species; the second ORF ( orf618), of 1854-bp, shares sequence similarity with pilS. The existence of the pilR-like and pilS-like genes in various X. campestris pathovars indicated that these two genes are well conserved in Xanthomonas. pilR and pilS mutants were constructed by gene replacement. We found that a pilR mutant, resistant to the infection of phage Cf, was unable to synthesize PilA protein; however, the abundance of the PilA protein and of the pilA transcript was markedly increased by the introduction of a plasmid containing the cloned pilR gene. The restoration of the normal pilus-specific sensitivity of this transformed clone to Cf indicated that the pilR gene functions as a transcriptional regulator of pilA. The pilS mutant, however, was susceptible to Cf infection, and the level of pilA expression in this mutant was similar to that of wild-type cells. Promoter analysis of luciferase reporter gene constructs containing the 5' untranslated regions of pilR or pilS genes revealed that, although the pilR and pilS are contiguous in X. a. pv. citri, the two genes are expressed independently, and the strong pilR promoter leads to the accumulation of PilR in X. a. pv. citri, which positively regulates the biosynthesis of PilA. These results revealed the enhanced sensitivity of X. a. pv. citri to phage Cf in the presence of PilR and indicated that the filamentous phage Cf utilize bacterial pili as a receptor site for its infection.

Amino Acid Sequence↗
PubMed1989

The nucleotide sequence around the capripoxvirus thymidine kinase gene reveals a gene shared specifically with leporipoxvirus.

We have extended previous comparisons of genetic organization between poxvirus genera by sequencing a 2.5K genomic fragment from isolate KS-1 (Kenya sheep-1) of the genus capripoxvirus. The fragment is located in the central region of the capripoxvirus genome and contains three complete and two incomplete open reading frames (ORFs). One of the complete ORFs is a gene for thymidine kinase (TK). This gene, with one of the other two complete ORFs and both the incomplete ORFs, are homologous to four contiguous ORFs from the central region of vaccinia virus (VV) DNA. They also match four ORFs of fowlpox virus (FPV) DNA, three of which are contiguous and the fourth, the FPV TK gene, is located elsewhere on the FPV genome. The third complete ORF of the capripoxvirus DNA fragment is located between the TK gene and the capripoxvirus homologue of the ORF immediately downstream of the VV TK gene. We show that a homologue to this third ORF is absent from VV and FPV DNAs, but is present downstream of the TK gene on Shope fibroma virus DNA. The sequence immediately upstream of the capripoxvirus homologue of a VV late gene contains a motif which is required for VV late gene expression. The motif required for VV early gene transcription termination is present in eight positions in the capripoxvirus sequence, and five of these positions are consistent with the motif having an equivalent function in capripoxvirus to that in VV.

Amino Acid Sequence↗
PubMed1992

Inducible expression bovine papillomavirus shuttle vectors containing ferritin translational regulatory elements.

The combination of transcriptional and translational control elements in an inducible expression vector suitable for use in stably transformed cell lines was explored. To this end, ferritin translational control elements have been inserted downstream from a mouse metallothionein (mMT-I) transcriptional promoter (PmMT-I), and upstream from various reporter protein-encoding open reading frames (ORFs), all carried on a bovine papillomavirus shuttle vector. Protocols which stimulate transcription (with zinc) and translation (with iron) were developed to optimize the induction of reporter protein synthesis. It was found that insertion of an iron regulatory element between the PmMT-I and a reporter ORF bestowed a sixfold inducibility of reporter protein synthesis with iron and a 90-fold inducibility with iron plus zinc in a classical superinduction protocol. Surprisingly, inclusion of other rabbit ferritin light chain sequences (rFL), including the ORF, enhanced reporter inducibilities to over 15- and 500-fold, respectively. These additional rFL sequences not only increased inducibility but also (i) increased the half-life of the mRNA and (ii) strongly inhibited translation of an ORF located downstream from the 5' proximal ORF. The maximum levels of reporter proteins attained in transformed cells after prolonged induction represented from 1% to 7% of total cellular protein. These inducible expression vectors should prove useful for the production and study of cytotoxic proteins.

Animals↗
PubMed1997

Baculovirus gp64 gene expression: negative regulation by a minicistron.

Small upstream open reading frames (ORFs) or minicistrons located in the 5' leader of eukaryotic mRNAs have been shown to play a role in translational regulation of some eukaryotic genes, particularly mammalian proto-oncogenes. A survey of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus genome suggests that at least 10 transcripts from late genes contain potential minicistrons, and at least three of these minicistrons appear to be conserved in homologous genes of the related Orygia pseudotsugata MNPV. The position of the minicistron from one of these genes, gp64, is also conserved in gp64 genes from several baculoviruses, suggesting a potential regulatory function. To identify the potential role of the gp64 minicistron in regulating translation from gp64 late mRNAs, we generated a series of recombinant viruses containing the gp64 promoter and minicistron in combination with a chloramphenicol acetyltransferase reporter gene (cat) inserted into the polyhedrin locus. We first fused a cat reporter in frame with the minicistron coding region to demonstrate that the minicistron initiator ATG was in a context suitable for translational initiation. In subsequent experiments, a cat reporter was fused in frame to the downstream gp64 ORF, and various constructs containing point mutations that inactivated the minicistron were examined. Translational efficiency in the presence and absence of the minicistron was measured by quantitative analysis of gp64-cat RNA and the GP64-CAT protein. In the absence of a functional minicistron, translational efficiency from the downstream gp64-cat reporter ORF increased. Surprisingly, single-point mutations that inactivated the minicistron initiator ATG also resulted in utilization of an upstream in-frame ATG that is found within the minicistron coding region and that is in a poor translational initiation context. Double-point mutation constructs that inactivated both the minicistron initiator ATG and the upstream in-frame ATG also resulted in increased translational efficiency from the downstream gp64-cat ORF. Thus, the gp64 minicistron serves as a negative regulatory element that decreases translation of the gp64 ORF on late mRNAs.

Animals↗
PubMed2001

Cloning, characterization, and possible origin of the Prevotella loescheii dnaK homolog.

Heat shock proteins play an important role in bacterial survival and response to environmental stress. We cloned the Prevotella loescheii HSP70 homolog (dnaK) and characterized the coding sequence, regulatory regions, and evolutionary relationships to other bacteria. Predicted proteins encoded by the P. loescheii dnaK homolog (open reading frame ORF-1) and two downstream coding regions, ORF-2 and ORF-3, are highly homologous to the proteins encoded by ORF-4 (dnaK), ORF-5, and ORF-6 from the dnaK region of Porphyromonas gingivalis. The dnaK promoter resembles other HSP (heat shock protein) promoters. Alignment of the predicted protein encoded by ORF-2 showed significant homology to the Bacteroides fragilis tnpA gene from the transposon Tn4555, whereas the ORF-3 protein showed homology to B. fragilis transposase (Tn5220) and integrase (Tn4555) proteins. This suggests a transposition-like event may be responsible for transfer of these genes between Porphyromonas and Prevotella.

Amino Acid Sequence↗
PubMed1988

Sequence and genetic organization of adenovirus type 35 early region 3.

The early transcription region 3 (E3) of group B adenovirus type 35 (Ad35), a serotype isolated primarily from patients with acquired immunodeficiency syndrome and other immunodeficiency disorders, has been partially sequenced. We had previously identified an Ad35 29-kilodalton (kDa) early glycoprotein which, analogous to group C Ad2 E3-19K, associated with major histocompatibility complex class I antigens in the endoplasmic reticulum of infected cells. The open reading frame (ORF) of the Ad35 29-kDa protein has now been identified within a 2-kilobase-pair cloned Ad35 E3 fragment. The predicted amino acid sequence was very similar to that of group B Ad3 E3-19K. In contrast, homology between the Ad35 and Ad2 glycoproteins was limited to five cysteines in identical positions and a 20-amino-acid region proximal to the transmembrane domain. In addition, 20.3- and 20.6-kDa ORFs have been identified downstream from the ORF for the Ad35 glycoprotein. Analogous 20-kDa ORFs are present in the Ad3 E3 region but are not present in Ad2 and Ad5. In contrast, the region analogous to an Ad2 11.6-kDa ORF, which is 9 kDa in size in Ad3, was absent from the expected position within the Ad35 E3 region. Because the E3 region is likely to play an important role in the interaction between virus and host, analysis of the function of the Ad35 E3 proteins should further our understanding of adenovirus pathogenesis.

Adenoviruses, Human↗
PubMed1998

Human T-cell leukemia virus type 1 reverse transcriptase (RT) originates from the pro and pol open reading frames and requires the presence of RT-RNase H (RH) and RT-RH-integrase proteins for its activity.

The first description of an active form of a recombinant human T-cell leukemia virus type 1 (HTLV-1) reverse transcriptase (RT) and subsequent predictions of its amino acid sequence and quaternary structure are reported here. By using amino acid alignment methods, the NH2 and COOH termini of the RT, RNase H (RH), and integrase (IN) domains of the Pol polyprotein were determined. The HTLV-1 RT seems to be unique since its NH2 terminus is probably encoded by the pro open reading frame (ORF) fused downstream, via a transframe peptide, to the polypeptide encoded by the pol ORF. The HTLV-1 Pol amino acid sequence was revealed to be highly similar to that of Rous sarcoma virus (RSV), particularly at the RT-RH hinge region. These two domains remain linked for RSV; this may also be the case for HTLV-1. In light of these results, RT, RT-RH, and RT-RH-IN genes were constructed and introduced into His-tagged protein expression vectors. The corresponding proteins were synthesized in vitro, and the DNA polymerase activities of different protein combinations were tested. Solely the RT-RH-RT-RH-IN combination was found to have a significant activity level. Velocity sedimentation analysis suggested that the HTLV-1 RT-RH and RT-RH-IN monomers are likely associated in an oligomeric structure, probably of the alpha3/beta type.

Amino Acid Sequence↗
PubMed1995

Function of a 5'-end genomic RNA mutation that evolves during persistent mouse hepatitis virus infection in vitro.

Persistently infected cultures of DBT cells were established with mouse hepatitis virus strain A59 (MHV-A59), and the evolution of the MHV leader RNA and 5' end of the genome was studied through 119 days postinfection. Sequence analysis of independent clones demonstrated an overall mutation frequency approaching 1.2 x 10(-3) to 6.7 x 10(-3). The rate of fixation of mutations was about 1.2 x 10(-5) to 7.6 x 10(-5) per nucleotide (nt) per day. In contrast to finding in bovine coronavirus, the MHV leader RNA sequences were extremely stable and did not evolve significantly during persistent infection. Rather, a 5' untranslated region (UTR) A-to-G mutation at nt 77 in the genomic RNA emerged by day 56 and accumulated until 50 to 80% of the genome-length molecules retained the mutation by 119 days postinfection. Although other 5'-end mutations were noted, only the nt 77 mutation was significantly associated with viral persistence in vitro. Mutations were also found in the 5' end of the p28 coding region, but no specific alterations accumulated in genome-length molecules through 119 days postinfection. The 5' UTR nt 77 mutation resulted in an 18-amino-acid open reading frame (ORF) upstream of the ORF 1a AUG start site. By in vitro translation assays, the small ORF was not translated into detectable product but the mutation significantly enhanced translation of the downstream p28 ORF about 2.5-fold. Variant viruses, containing either the nt 77 A-to-G mutation (V16-ATG+) or wild-type sequences at this locus (V1-ATG-), were isolated at 119 days postinfection. The variant viruses replicated more efficiently than wild-type virus and were extremely cytolytic in DBT cells, suggesting that the A-to-G mutation did not encode a nonlytic or attenuated phenotype. Consistent with the in vitro translation results, a significant increase (approximately 3.5-fold) in p28 expression was also observed with the mutant virus (V16-ATG+) in DBT cells compared with that in wild-type controls. These data indicate that MHV persistence was significantly associated with mutation and evolution in the 5'-end UTR which enhanced the translation of the ORF 1a and potentially ORF 1b polyproteins which function in virus transcription and replication.

Amino Acid Sequence↗
PubMed2004

Short-range compositional correlation in the yeast genome depends on transcriptional orientation.

This article reports an analysis of composition of about 5000 intergenic regions and neighboring ORFs in the nuclear genome of Saccharomyces cerevisiae, and their correlation. Intergenic regions flanked by divergently transcribed ORFs are GC richer (36%) than those separating convergent ORFs (29%). This difference in GC content cannot be fully attributed to its location upstream or downstream the ORFs, since no such strong compositional bias is found within 3' and 5' segments of intergenic regions between ORFs transcribed in the same direction. We have also found that the GC content of intergenic regions is positively correlated to that of its flanking ORFs in tandem and divergent orientations, but not in convergent orientations, and that the correlation coefficient between the GC content of nearby ORFs is higher for divergent pairs. Our observations are discussed in the light of recent work stressing the relationships between base composition, chromatin structure and meiotic recombination.

Base Composition↗
PubMed1997

TRAP transporters: a new family of periplasmic solute transport systems encoded by the dctPQM genes of Rhodobacter capsulatus and by homologs in diverse gram-negative bacteria.

The dct locus of Rhodobacter capsulatus encodes a high-affinity transport system for the C4-dicarboxylates malate, succinate, and fumarate. The nucleotide sequence of the region downstream of the previously sequenced dctP gene (encoding a periplasmic C4-dicarboxylate-binding protein) was determined. Two open reading frames (ORFs) of 681 bp (dctQ) and 1,320 bp (dctM) were identified as additional dct genes by insertional mutagenesis and complementation studies. DctQ (24,763 Da) and DctM (46,827 Da) had hydropathic profiles consistent with the presence of 4 and 12 potential transmembrane segments, respectively, and were localized in the cytoplasmic membrane fraction after heterologous expression of the dctQM ORFs in Escherichia coli. DctP, DctQ, and DctM were found to be unrelated to known transport proteins in the ABC (ATP-binding cassette) superfamily but were shown to be homologous with the products of previously unidentified ORFs in a number of gram-negative bacteria, including Bordetella pertussis, E. coli, Salmonella typhimurium, Haemophilus influenzae, and Synechocystis sp. strain PCC6803. An additional ORF (rypA) downstream of dctM encodes a protein with sequence similarity to eukaryotic protein-tyrosine phosphatases, but interposon mutagenesis of this ORF did not result in a Dct- phenotype. Complementation of a Rhizobium meliloti dctABD deletion mutant by heterologous expression of the dctPQM genes from R. capsulatus demonstrated that no additional structural genes were required to form a functional transport system. Transport via the Dct system was vanadate insensitive, and in uncoupler titrations with intact cells, the decrease in the rate of succinate transport correlated closely with the fall in membrane potential but not with the cellular ATP concentration, implying that the proton motive force, rather than ATP hydrolysis, drives uptake. It is concluded that the R. capsulatus Dct system is a new type of periplasmic secondary transporter and that similar, hitherto-unrecognized systems are widespread in gram-negative bacteria. The name TRAP (for tripartite ATP-independent periplasmic) transporters is proposed for this new group.

Adenosine Triphosphate↗
PubMed2004

Splicing of Cauliflower mosaic virus 35S RNA serves to downregulate a toxic gene product.

Alternative splicing usually leads to an increase in the number of gene products that can be derived from a single transcript. Here, a different and novel use of alternative splicing--as a means to control the amount of a potentially toxic gene product in the plant pararetrovirus Cauliflower mosaic virus (CaMV)--is reported. About 70 % of the CaMV 35S RNA, which serves as a substrate for both reverse transcription and polycistronic mRNA, is spliced into four additional RNA species. Splicing occurs between four donor sites--one in the 5' untranslated region and three within open reading frame (ORF) I--and one unique acceptor site at position 1508 in ORF II. A previous study revealed that the acceptor site is vital for CaMV infectivity and expression of ORFs III and IV from one of the spliced RNA species suggested that splicing may facilitate expression of downstream CaMV ORFs. However, it is shown here that deleting the splice acceptor site and replacing ORF II with a cargo ORF that lacks splice acceptor sites does not interfere with virus proliferation. Furthermore, it is demonstrated that whenever P2 cannot accumulate in infected tissues, the splice acceptor site at position 1508 is no longer vital and has little effect on virus replication. This suggests that the vital role of splicing in CaMV is regulation of P2 expression and that P2 exhibits biological properties that, whilst indispensable for virus-vector interactions, can block in planta virus infection if this regulation is abolished.

5' Untranslated Regions↗
PubMed1997

Cell-density-dependent expression of Borrelia burgdorferi lipoproteins in vitro.

Previously, we had identified non-OspA-OspB surface proteins of Borrelia burgdorferi that are targeted by the antibody-dependent complement-mediated killing mechanism. Here we demonstrate by Western blotting that one of these proteins, P35, is upregulated at the onset of stationary phase in vitro. Northern analysis revealed that the upregulation of P35 is at the level of transcription. In addition, the expression of an open reading frame (ORF) located downstream of the p35 gene was found to be regulated in the same fashion as that of P35. This ORF encodes a 7.5-kDa lipoprotein. The transcriptional start sites for both of these genes were determined, to aid in the identification of the putative promoter regions. Additional sequencing of the 5' flanking region of the p35 gene revealed a region of dyad symmetry 52 bp upstream of the transcription start site. Southern analysis demonstrated that the expression of these genes was not due to a cell-density-dependent rearrangement in the genome of B. burgdorferi. These findings provide an in vitro model for studying mechanisms of gene regulation in B. burgdorferi.

Amino Acid Sequence↗
PubMed1998

Several new bacteriophage T4 genes, mapped by sequencing deletion endpoints between genes 56 (dCTPase) and dda (a DNA-dependent ATPase-helicase) modulate transcription.

We have analyzed DNA of wild-type T4 and of 13 independent large viable deletions isolated by Homyk and Weil (Virology 61 (1974) 505-523) and by Little (Virology 53 (1973) 47-59), by sequencing, cloning, and expression studies. The deletions can be explained by illegitimate recombination between short (4- to 15-bp) ectopic repeats. In four deletions, adjacent regions are partially homologous, and in at least one of them, the base adjacent to the overlap was deleted during recombination. The sequence 5'-GGGC, which has not been associated with T4 deletions in other map regions, occurs within three repeats, and near the repeats in four more of the 13 deletions. Five previously named genes, 69, soc, mrh, modA, and dda were mapped relative to the deletion endpoints. Nine additional ORFs were found interspersed between them. One of these shares some similarities with mrh (modulates rpoH; Frazier and Mosig, Gene 88 (1990) 7-14), and another one resembles modA (coding for an ADP-ribosyl-transferase that modifies RNA polymerase alpha subunits, Skórko et al., Eur. J. Biochem. 79 (1977) 55-66) respectively. We found that the host's heat shock sigma factor, sigma32, is phosphorylated, and that Mrh protein modulates this phosphorylation. The ORF dda.9 downstream of mrh has a patchy similarity with conserved C-terminal segments (motifs) of sigma32; therefore, we call it srh. Another ORF, dda.2 located between modA and dda, shares sequence similarity with sigma70, and we call it srd. We consider the possibility that Srh and Srd act as decoys for sigma32, or sigma70, respectively. Expression of several of the ORFs from cloned DNA appears to be toxic to the host bacteria. Mutant clones only could be constructed from gene 69 and from modA. Moreover, dda.2 (srd)-containing bacteria grow extremely slowly, and they form filaments in liquid cultures. Clones carrying mrh and srh show less severe filamentation. Our results highlight the importance of 'non-essential' genes for phage development and evolution.

Amino Acid Sequence↗
PubMed1990

Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli.

The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.

Alkyl and Aryl Transferases↗
PubMed2002

Functional and mutational analysis of conjugative transfer region 1 (Tra1) from the IncHI1 plasmid R27.

The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region between traH and trhR was cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of the oriT region are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of the oriT region are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.

Base Sequence↗