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PubMed2004

A domain interaction map based on phylogenetic profiling.

Phylogenetic profiling is a well established method for predicting functional relations and physical interactions between proteins. We present a new method for finding such relations based on phylogenetic profiling of conserved domains rather than proteins, avoiding computationally expensive all versus all sequence comparisons among genomes. The resulting domain interaction map (DIMA) can be explored directly or mapped to a genome of interest. We demonstrate that the performance of DIMA is comparable to that of classical phylogenetic profiling and its predictions often yield information that cannot be detected by profiling of entire protein chains. We provide a list of novel domain associations predicted by our method.

Cluster Analysis↗
PubMed1992

ToxR proteins with substitutions in residues conserved with OmpR fail to activate transcription from the cholera toxin promoter.

The ToxR protein of Vibrio cholerae is an integral membrane protein that coordinately regulates the expression of virulence genes required for successful infection. ToxR has been shown to bind directly to and activate transcription of the cholera toxin (ctx) promoter. Within the amino-terminal cytoplasmic region of ToxR, several amino acids are strictly conserved among ToxR, OmpR, and the other members of a family of bacterial regulatory proteins. To better understand the function of this region, two approaches were taken: conserved residues were changed by site-directed mutagenesis, and random mutations that eliminated ToxR-mediated transcriptional activation were isolated. Several classes of mutations were identified: those that abolish promoter DNA binding and transcriptional activation (toxR R96K, toxR R68K, and toxR R68L), those that abolish transcriptional activation but retain the ability to bind promoter DNA (toxR R96L), and those that have an intermediate phenotype (toxR R77L, toxR E51K, and toxR E51D). The toxR E51K allele had reduced activity in both Escherichia coli and V. cholerae but also exerted a dominant-negative effect over wild-type ToxR when assayed in V. cholerae. This result provides additional evidence that ToxR acts as an oligomer in the transcriptional activation process. From this mutational analysis of conserved amino acid residues within the OmpR-homologous region of ToxR, we conclude that this region is essential for transcriptional activation at the level of DNA binding and other steps that lead to activation of the ctx promoter.

Amino Acid Sequence↗
PubMed2004

The Evolution of SINEs and LINEs in the genus Chironomus (Diptera).

Genomic DNA amplification from 51 species of the family Chironomidae shows that most contain relatives of NLRCth1 LINE and CTRT1 SINE retrotransposons first found in Chironomus thummi. More than 300 cloned PCR products were sequenced. The amplified region of the reverse transcriptase gene in the LINEs is intact and highly conserved, suggesting active elements. The SINEs are less conserved, consistent with minimal/no selection after transposition. A mitochondrial gene phylogeny resolves the Chironomus genus into six lineages (Guryev et al. 2001). LINE and SINE phylogenies resolve five of these lineages, indicating their monophyletic origin and vertical inheritance. However, both the LINE and the SINE tree topologies differ from the species phylogeny, resolving the elements into "clusters I-IV" and "cluster V" families. The data suggest a descent of all LINE and SINE subfamilies from two major families. Based on the species phylogeny, a few LINEs and a larger number of SINEs are cladisitically misplaced. Most misbranch with LINEs or SINEs from species with the same families of elements. From sequence comparisons, cladistically misplaced LINEs and several misplaced SINEs arose by convergent base substitutions. More diverged SINEs result from early transposition and some are derived from multiple source SINEs in the same species. SINEs from two species (C. dorsalis, C. pallidivittatus), expected to belong to the clusters I-IV family, branch instead with cluster V family SINEs; apparently both families predate separation of cluster V from clusters I-IV species. Correlation of the distribution of active SINEs and LINEs, as well as similar 3' sequence motifs in CTRT1 and NLRCth1, suggests coevolving retrotransposon pairs in which CTRT1 transposition depends on enzymes active during NLRCth1 LINE mobility.

Animals↗
PubMed1992

Mutational analysis of signal transduction by ArcB, a membrane sensor protein responsible for anaerobic repression of operons involved in the central aerobic pathways in Escherichia coli.

In Escherichia coli, the expression of a group of operons involved in aerobic metabolism is regulated by a two-component signal transduction system in which the arcB gene specifies the membrane sensor protein and the arcA gene specifies the cytoplasmic regulator protein. ArcB is a large protein belonging to a subclass of sensors that have both a transmitter domain (on the N-terminal side) and a receiver domain (on the C-terminal side). In this study, we explored the essential structural features of ArcB by using mutant analysis. The conserved His-292 in the transmitter domain is indispensable, indicating that this residue is the autophosphorylation site, as shown for other homologous sensor proteins. Compression of the range of respiratory control resulting from deletion of the receiver domain and the importance of the conserved Asp-533 and Asp-576 therein suggest that the domain has a kinetic regulatory role in ArcB. There is no evidence that the receiver domain enhances the specificity of signal transduction by ArcB. The defective phenotype of all arcB mutants was corrected by the presence of the wild-type gene. We also showed that the expression of the gene itself is not under respiratory regulation.

Amino Acid Sequence↗
PubMed2006

Genetic structure and assignment tests demonstrate illegal translocation of red deer (Cervus elaphus) into a continuous population.

Molecular forensic methods are being increasingly used to help enforce wildlife conservation laws. Using multilocus genotyping, illegal translocation of an animal can be demonstrated by excluding all potential source populations as an individual's population of origin. Here, we illustrate how this approach can be applied to a large continuous population by defining the population genetic structure and excluding suspect animals from each identified cluster. We aimed to test the hypothesis that recreational hunters had illegally introduced a group of red deer into a hunting area in Luxembourg. Reference samples were collected over a large area in order to test the possibility that the suspect individuals might be recent immigrants. Due to isolation-by-distance relationships in the data set, inferring the number of genetic clusters using Bayesian methods was not straightforward. Biologically meaningful clusters were only obtained by simultaneously analysing spatial and genetic information using the program baps 4.1. We inferred the presence of three genetic clusters in the study region. Using partial Mantel tests, we detected barriers to gene flow other than distance, probably created by a combination of urban areas, motorways and a river valley used for viticulture. The four focal animals could be excluded with a high certainty from the three genetic subpopulations and it was therefore likely that they had been released illegally.

Animal Migration↗
PubMed2007

Leishmania donovani bisubunit topoisomerase I gene fusion leads to an active enzyme with conserved type IB enzyme function.

All eukaryotic topoisomerase I enzymes are monomeric enzymes, whereas the kinetoplastid family (Trypanosoma and Leishmania) possess an unusual bisubunit topoisomerase I. To determine what happens to the enzyme architecture and catalytic property if the two subunits are fused, and to explore the functional relationship between the two subunits, we describe here in vitro gene fusion of Leishmania bisubunit topoisomerase I into a single ORF encoding a new monomeric topoisomerase I (LdTOPIL-fus-S). It was found that LdTOPIL-fus-S is active. Gene fusion leads to a significant modulation of in vitro topoisomerase I activity compared to the wild-type heterodimeric enzyme (LdTOPILS). Interestingly, an N-terminal truncation mutant (1-210 amino acids) of the small subunit, when fused to the intact large subunit [LdTOPIL-fus-Delta(1-210)S], showed reduced topoisomerase I activity and camptothecin sensitivity in comparison to LdTOPIL-fus-S. Investigation of the reduction in enzyme activity indicated that the nonconserved 1-210 residues of LdTOPIS probably act as a 'pseudolinker' domain between the core and catalytic domain of the fused Leishmania enzyme, whereas mutational analysis of conserved His453 in the core DNA-binding domain (LdTOPIL) strongly suggested that its role is to stabilize the enzyme-DNA transition state through hydrogen bonding to one of the nonbridging oxygens. Taken together, our findings provide an insight into the details of the unusual structure of bisubunit topoisomerase I of Leishmania donovani.

Animals↗
PubMed2002

Deciphering endocytosis in Caenorhabditis elegans.

We discuss in this review recent studies using the worm Caenorhabditis elegans to decipher endocytic trafficking in a multicellular organism. Recent advances, including in vivo assay systems, new genetic screens, comparative functional analysis of conserved proteins, and RNA-mediated interference (RNAi) in C. elegans, are being used to study the functions of known membrane trafficking factors and to identify new ones. The ability to monitor endocytosis in vivo in worms allows one to test current endocytosis models and to demonstrate the physiological significance of factors identified by genetic and biochemical methods. The available human genome sequence facilitates comparative studies where human homologs of new factors identified in C. elegans can be quickly assayed for similar function using traditional cell biological methods in mammalian cell systems. New studies in C. elegans have used a combination of these techniques to reveal novel metazoan-specific trafficking factors required for endocytosis. Many more metazoan-specific trafficking factors and insights into the mechanisms of endocytosis are likely to be uncovered by analysis in C. elegans.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Characterization of 23 CC chemokine genes and analysis of their expression in channel catfish (Ictalurus punctatus).

Chemokines are a large family of chemotactic cytokines playing crucial roles in the innate immune response. CC chemokines constitute the largest subfamily of chemokines, with 28 CC chemokines identified from mammalian species. However, the status of CC chemokines in teleosts is yet to be determined. We previously identified 26 catfish CC chemokine cDNAs from catfish. In this study, we isolated and sequenced 23 channel catfish CC chemokine genes amounting to a total of over 56 kb of genomic sequences. Genomic organization of the 23 CC chemokine genes was determined by comparing the generated genomic sequences with the previously identified cDNA sequences. Microsatellites were identified from 16 catfish CC chemokine genes allowing them to be utilized for genome mapping. Structural analysis indicated conservation of genomic organization of CC chemokine genes, which may facilitate the establishment of orthologies. Expression of all known catfish CC chemokine transcripts was assessed in nine important tissues. Of the 26 catfish CC chemokine genes, 14 were universally expressed, six were widely expressed in many tissues, while six were highly tissue-specific.

Amino Acid Sequence↗
PubMed2001

Variability of the N-protein and the intergenic region of the S RNA of tomato spotted wilt tospovirus (TSWV).

The genetic heterogeneity of the N protein gene and the intergenic region (IGR) of the S RNA from tomato spotted wilt virus (TSWV) isolates, collected in Bulgaria, were compared with isolates from other parts of the world. The results substantiated the highly conserved nature of the N protein. Twenty six independent sequences revealed only seven variable amino acid positions, common to all isolates. The type of amino acids present in these positions seems to be independent of the geographical origin. In contrast to the structural N protein, comparisons of the related IGR-sequences led to clusters correlated with the geographical origin of the isolates. Although the overall sequence homology in the IGRs was much lower than for the N proteins, three conserved parts within this region were identified. The outstanding part was a central area of 31 nucleotides with a significantly increased GC-content. This was located in both viral- and viral-complement RNA at structures with similar foldings, which led to the assumption that this stabilised structure, rather than a sequence motive, might serve as a transcription terminator during the synthesis of the two mRNAs from the ambisense segments.

Amino Acid Sequence↗
PubMed2003

Cloning and expression of Blo t 1, a novel allergen from the dust mite Blomia tropicalis, homologous to cysteine proteases.

BACKGROUND: House dust mite allergens have been shown to be a very important stimulus in the causation of asthma and triggers for the exacerbation of symptoms. Therefore, characterization of mite-derived allergens at the molecular level is an important step for the development of effective diagnostic and therapeutic approaches, as well as for epidemiological studies. OBJECTIVE: To clone, express and characterize at the molecular level the cysteine protease from Blomia tropicalis (Bt). METHODS: A full-length cDNA encoding Blo t 1 was cloned from a Bt cDNA library using a PCR and RACE-based strategy. The cDNA was PCR-amplified, sequenced and subcloned into a prokaryotic expression vector. The allergenicity of the recombinant Blo t 1 was evaluated for IgE reactivity by Western blot. RESULTS: Blo t 1 cDNA encodes a 221 amino acids polypeptide with an estimated molecular weight of 25 kDa. The recombinant protein is 35% identical to other mite cysteine proteases. Recombinant Blo t 1 (rBlo t 1) bound IgE from 62% of Bt skin test-positive serum. Dermatophagoides pteronyssinus (Dp) skin test-positive sera did not react with rBlo t 1, indicating the possible presence of unique IgE epitopes on the rBlo t 1 molecule. A three-dimensional image of Blo t 1, constructed based on predicted analysis, showed conserved secondary and tertiary structure with other cysteine proteases. CONCLUSION: We report the cloning, expression and IgE reactivity of Blo t 1, a novel allergen from the domestic mite Blomia tropicalis (Bt), highly homologous to cysteine proteases. This putative cysteine protease, designated Blo t 1, may play a major role as an immunodominant allergen involved in dust mite-specific IgE-mediated hypersensitivity.

Allergens↗
PubMed2002

A functionally conserved member of the FTZ-F1 nuclear receptor family from Schistosoma mansoni.

The fushi tarazu factor 1 (FTZ-F1) nuclear receptor subfamily comprises orphan receptors with crucial roles in development and sexual differentiation in vertebrates and invertebrates. We describe the structure and functional properties of an FTZ-F1 from the platyhelminth parasite of humans, Schistosoma mansoni, the first receptor from this family to be characterized in a Lophotrochozoan. It contains a well conserved DNA-binding domain (55-63% identity to other family members) and a poorly conserved ligand-binding domain (20% identity to that of zebrafish FF1a). However, both the ligand domain signature sequence and the activation function 2-activation domain (AF2-AD) are perfectly conserved. Phylogenetic analysis confirmed that SmFTZ-F1 is a member of nuclear receptor subfamily 5, but that it clustered with the Drosophila receptor DHR39 and has consequently been named NR5B1. The gene showed a complex structure with 10 exons and an overall size of 18.4 kb. Two major transcripts were detected, involving alternative promoter usage and splicing of the two 5' exons, but which encoded identical proteins. SmFTZ-F1 mRNA is expressed at all life-cycle stages with the highest amounts in the larval forms (miracidia, sporocysts and cercariae). However, expression of the protein showed a different pattern; low in miracidia and higher in adult male worms. The protein bound the same monomeric response element as mammalian SF-1 (SF-1 response element, SFRE) and competition experiments with mutant SFREs showed that its specificity was identical. Moreover, SmFTZ-F1 transactivated reporter gene transcription from SFRE similarly to SF-1. This functional conservation argues for a conserved biological role of the FTZ-F1 nuclear receptor family throughout the metazoa.

Amino Acid Sequence↗
PubMed2000

Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate.

Peroxidases of the peroxiredoxin (Prx) family contain a Cys residue that is preceded by a conserved sequence in the NH(2)-terminal region. A new type of mammalian Prx, designated PrxV, has now been identified as the result of a data base search with this conserved Cys-containing sequence. The 162-amino acid PrxV shares only approximately 10% sequence identity with previously identified mammalian Prx enzymes and contains Cys residues at positions 73 and 152 in addition to that (Cys(48)) corresponding to the conserved Cys. Analysis of mutant human PrxV proteins in which each of these three Cys residues was individually replaced with serine suggested that the sulfhydryl group of Cys(48) is the site of oxidation by peroxides and that oxidized Cys(48) reacts with the sulfhydryl group of Cys(152) to form an intramolecular disulfide linkage. The oxidized intermediate of PrxV is thus distinct from those of other Prx enzymes, which form either an intermolecular disulfide or a sulfenic acid intermediate. The disulfide formed by PrxV is reduced by thioredoxin but not by glutaredoxin or glutathione. Thus, PrxV mutants lacking Cys(48) or Cys(152) showed no detectable thioredoxin-dependent peroxidase activity, whereas mutation of Cys(73) had no effect on activity. Immunoblot analysis revealed that PrxV is widely expressed in rat tissues and cultured mammalian cells and is localized intracellularly to cytosol, mitochondria, and peroxisomes. The peroxidase function of PrxV in vivo was demonstrated by the observations that transient expression of the wild-type protein, but not that of the Cys(48) mutant, in NIH 3T3 cells inhibited H(2)O(2) accumulation and activation of c-Jun NH(2)-terminal kinase induced by tumor necrosis factor-alpha.

Amino Acid Sequence↗
PubMed2001

Improving outcomes in acute coronary syndromes--the FRISC II trial.

The FRISC II study addressed two key questions in the management of acute coronary syndromes: is it beneficial to extend low-molecular-weight heparin (LMWH) therapy with dalteparin beyond the initial period of acute treatment; and, is a strategy of early invasive therapy, including angioplasty and surgical revascularization, preferable to a more conservative strategy? The study focused on patients with unstable coronary artery disease (UCAD), that is, angina and non-ST-segment-elevation myocardial infarction (MI). Patients were allocated in a randomized, factorial study design to either an invasive or a conservative management strategy. Within each of these groups, patients were further randomized to receive either 3 months of extended treatment with dalteparin or placebo, following at least 5 days' treatment with open-label dalteparin. After 1 year, patient survival and MI-free survival were significantly higher in the invasive therapy group than in the noninvasive group. Patients who received extended dalteparin treatment had a significantly reduced probability of death or MI after 1 month (relative risk reduction 47%; p = 0.002), a benefit still evident after 60 days, but after 3 months there was no longer any significant clinical advantage compared with placebo. There was, however, a significant reduction in the combined incidence of death, MI, or revascularization at 3 months in the extended dalteparin treatment group (relative risk reduction 13%; p = 0.031). The benefits of extended dalteparin treatment were particularly marked in patients with elevated troponin-T or ST-segment depression. A subgroup analysis of conservatively managed patients who underwent revascularization in the first 45 days revealed that the probability of death or MI at 1 year was significantly lower among patients who received extended dalteparin treatment (relative risk reduction 35%; p = 0.02). Extended dalteparin treatment is, however, associated with a small increase in bleeding risk. In conclusion, early invasive therapy (following combined treatment with aspirin and dalteparin) is recommended in a majority of patients with UCAD. Furthermore, extended dalteparin treatment for up to 45 days is efficacious and well tolerated, and therefore provides a useful "bridge" to revascularization when early revascularization is not immediately available.

Adult↗
PubMed1993

Complete sequence conservation of the human T cell leukaemia virus type 1 tax gene within a family cluster showing different pathologies.

We have amplified, through PCR, the full-length tax gene of human T cell leukaemia virus type 1 (HTLV-1) derived from proviral DNA of peripheral blood lymphocytes of five first degree relatives of Afro-Caribbean origin. One patient (the father) had adult T cell leukaemia (ATL), one (the mother) tropical spastic paraparesis (TSP), and three (children) were healthy asymptomatic carriers. All five family members had identical tax nucleotide sequences as determined by direct sequencing of PCR products. This sequence was compared with tax gene sequences of an unrelated TSP patient of Afro-Caribbean origin, and of C8166 cells, and found to have one and seven nucleotide differences, respectively. At the amino acid level these three sequences differed from the HTLV-1 prototype Japanese strain (ATK-1). All sequence changes were clustered towards the 3' end of the gene. These data demonstrate the complete conservation of an HTLV-1 gene following, presumably, horizontal and vertical transmission of the virus. Clones of this gene showed more sequence variation within the TSP patient than the ATL patient, mostly consisting of point mutations; there was no conservation of mutations between the two individuals. These mutations occurred only in individual clones of the ATL patient whereas those of the TSP patient were found to be repeated in different clones. A tax-specific cytotoxic T lymphocyte response was observed in two asymptomatic carriers with low antibody titres, whereas none was detected in an individual with a high antibody level. No tax-specific sequence was identified which may have contributed to the apparently high degree of transmission from mother to children (three of five children tested) nor account for the differences between disease symptoms in the parents.

Adult↗
PubMed2003

Polymorphisms of interleukin-1alpha constitute independent risk factors for chronic graft-versus-host disease after allogeneic bone marrow transplantation.

The interleukin-1 (IL-1) family of cytokines is widely involved in inflammatory processes and diseases with an inflammatory component. Polymorphisms of the IL-1alpha, IL-1beta and IL-1Ra genes have been implicated in a number of autoimmune or inflammatory conditions, with polymorphism of the IL-1Ra gene showing association with severity of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). We compared the clinical outcomes (GVHD and survival) of 115 patients after human leucocyte antigen (HLA)-identical sibling allogeneic BMT with their genotype for two polymorphisms present in the IL-1alpha gene, which have been implicated in immune-related pathology. Possession of allele 2 of the IL-1alpha-889 polymorphism and allele 2 of the IL-1alpha variable number tandem repeat (VNTR) polymorphism in the donor genotype was associated with the occurrence of chronic, but not acute GVHD. A local normal population was also genotyped for these polymorphisms, and subsequent analysis identified conserved haplotypes in this gene region. Haplotypes containing allele 2 at both IL-1alpha-889 and IL-1alpha VNTR loci were extremely uncommon, suggesting that both risk alleles would be inherited independently. Both loci could therefore function as independent disease association markers. The polymorphisms of the IL-1alpha gene could be used to predict chronic GVHD in HLA-matched sibling transplants alongside clinical risk factors.

Adult↗
PubMed1986

In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for "extended" promoter elements in gram-positive organisms.

Analysis of Clostridium pasteurianum genomic DNA indicates that the ferredoxin (Fd) gene is present in a single copy. The cloned Fd gene previously described (Graves, M.C., Mullenbach, G. T., and Rabinowitz, J. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1653-1657) was used to map in vivo and in vitro synthesized Fd transcripts. The in vivo mRNA was sized in two ways: by Northern hybridization analysis, and more directly from the known DNA sequence after the 5'- and 3'-termini were identified. The 5'-end was determined by primer extension-dideoxy sequencing and the 3'-end by S1 nuclease mapping. The monocistronic Fd mRNA contains about 255 nucleotides and, thus, is one of the shortest bacterial mRNAs yet described. We also examined the Fd transcripts produced by Escherichia coli transformed with the plasmid containing the Fd gene. E. coli RNA polymerase most likely recognizes the same promoter (P1) as the clostridial polymerase, and furthermore, efficiently uses an additional promoter (P2) that is poorly recognized by the normal host enzyme. For comparison, in vitro transcripts were generated by E. coli and Bacillus subtilis RNA polymerases. In vitro, only promoter P1 is used by either E. coli or B. subtilis RNA polymerase. The 3'-end of each of the four types of transcripts occurs essentially at the same location and maps to within a large dyad symmetry element. Comparison of the Fd promoter with other Gram-positive promoters reveals that some sequences outside of the traditional Pribnow and -35 regions are conserved. This analysis indicates that an "extended" promoter recognition site may be required in these organisms.

Base Sequence↗
PubMed2006

Micromonas pusilla reovirus: a new member of the family Reoviridae assigned to a novel proposed genus (Mimoreovirus).

Micromonas pusilla reovirus (MpRV) is an 11-segmented, double-stranded RNA virus isolated from the marine protist Micromonas pusilla. Sequence analysis (including conserved termini and presence of core motifs of reovirus polymerase), morphology and physicochemical properties confirmed the status of MpRV as a member of the family Reoviridae. Electron microscopy showed that intact virus particles are unusually larger (90-95 nm) than the known size of particles of viruses belonging to the family Reoviridae. Particles that were purified on caesium chloride gradients had a mean size of 75 nm (a size similar to the size of intact particles of members of the family Reoviridae), indicating that they lost outer-coat components. The subcore particles had a mean size of 50 nm and a smooth surface, indicating that MpRV belongs to the non-turreted Reoviridae. The maximum amino acid identity with other reovirus proteins was 21 %, which is compatible with values existing between distinct genera. Based on morphological and sequence findings, this virus should be classified as the representative of a novel genus within the family Reoviridae, designated Mimoreovirus (from Micromonas pusilla reovirus). The topology of the phylogenetic tree built with putative polymerase sequences of the family Reoviridae suggested that the branch of MpRV could be ancestral. Further analysis showed that segment 1 of MpRV was much longer (5792 bp) than any other reovirus segment and encoded a protein of 200 kDa (VP1). This protein exhibited significant similarities to O-glycosylated proteins, including viral envelope proteins, and is likely to represent the additional outer coat of MpRV.

Amino Acid Sequence↗
PubMed1995

Confirmation of the helical structure of the 5'/3' termini of the essential DNA packaging pRNA of phage phi 29.

Bacteriophage phi 29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during viral assembly. An intriguing feature of phi 29 is the presence of a 120-base virus-encoded RNA (pRNA) that is indispensable for DNA packaging. Phylogenetic comparison of similar RNAs in numerous phages has revealed that the secondary structure of the pRNA is well conserved. Computer analysis predicts the presence of an extensive segment of helix with three single-base bulges generated by the pairing of the 5' and 3' ends. The desire to understand the role played by the pRNA in DNA packaging has led to a mutational analysis of the 5'-/3'-terminal region, which is believed to be important in DNA translocation. Deletion of 3 bases from the 3' end of the RNA, shortening the pRNA from 120 to 117 bases, was tolerated without loss of activity, but additional deletion of the base 117 resulted in 100-fold less activity, and a 115-base pRNA was virtually nonfunctional. Additionally, the three unpaired one-base bulges within the helical stretches of the paired proximate ends were nonessential for pRNA activity, as demonstrated by deletion of the bulge individually. An extensive series of helix disruptions by single- and multiple-base substitution almost invariably led to the loss of DNA packaging activity. Additional mutations that restored predicted base pairings rescued pRNA activity. This second site suppression confirmed that the 5'- and 3'-end region was paired and was indeed a helical stretch. The secondary structure was of greater importance than the primary sequence, with the exception of the requirement of an adenine at either the third or fourth position. The specific requirement of an adenine in phi 29 pRNA at this position, as well as conservation of this position in other phage pRNAs, implicates that this base may play a special role in either the DNA-packaging reaction or the maintenance of the pRNA tertiary structure.

Bacillus Phages↗