Non-conserved Sequences Research Articles

Interaction of the U1 snRNP with nonconserved intronic sequences affects 5' splice site selection.

Intron definition and splice site selection occur at an early stage during assembly of the spliceosome, the complex mediating pre-mRNA splicing. Association of U1 snRNP with the pre-mRNA is required for these early steps. We report here that the yeast U1 snRNP-specific protein Nam8p is a component of the commitment complexes, the first stable complexes assembled on pre-mRNA. In vitro and in vivo, Nam8p becomes indispensable for efficient 5' splice site recognition when this process is impaired as a result of the presence of noncanonical 5' splice sites or the absence of a cap structure. Nam8p stabilizes commitment complexes in the latter conditions. Consistent with this, Nam8p interacts with the pre-mRNA downstream of the 5' splice site, in a region of nonconserved sequence. Substitutions in this region affect splicing efficiency and alternative splice site choice in a Nam8p-dependent manner. Therefore, Nam8p is involved in a novel mechanism by which a snRNP component can affect splice site choice and regulate intron removal through its interaction with a nonconserved sequence. This supports a model where early 5' splice recognition results from a network of interactions established by the splicing machinery with various regions of the pre-mRNA.

Solution structure of potassium channel‐inhibiting scorpion toxin Lq2

Lq2 is a unique scorpion toxin. Acting from the extracellular side, Lq2 blocks the ion conduction pore in not only the voltage- and Ca2+-activated channels, but also the inward-rectifier K+ channels. This finding argues that the three-dimensional structures of the pores in these K+ channels are similar. However, the amino acid sequences that form the external part of the pore are minimally conserved among the various classes of K+ channels. Because Lq2 can bind to all the three classes of K+ channels, we can use Lq2 as a structural probe to examine how the non-conserved pore-forming sequences are arranged in space to form similar pore structures. In the present study, we determined the three-dimensional structure of Lq2 using nuclear magnetic resonance (NMR) techniques. Lq2 consists of an α-helix (residues S10 to L20) and a β-sheet, connected by an αβ3 loop (residues N22 to N24). The β-sheet has two well-defined anti-parallel strands (residues G26 to M29 and residues K32 to C35), which are connected by a type I' β-turn centered between residues N30 and K31. The N-terminal segment (residues Z1 to T8) appears to form a quasi-third strand of the β-sheet. Proteins 1999;34:417–426. © 1999 Wiley-Liss, Inc.

Highly conserved RNA sequences that are sensors of environmental stress.

The putative function of highly conserved regions (HCRs) within 3' untranslated regions (3'UTRs) as regulatory RNA sequences was efficiently and quantitatively assessed by using modular retroviral vectors. This strategy led to the identification of HCRs that alter gene expression in response to oxidative or mitogenic stress. Databases were screened for UTR sequences of >100 nucleotides that had retained 70% identity over more than 300 million years of evolution. The effects of 10 such HCRs on a standard reporter mRNA or protein were studied. To this end, we developed a modular retroviral vector that can allow for a direct comparison of the effects of different HCRs on gene expression independent of their gene-intrinsic 5'UTR, promoter, protein coding region, or poly(A) sequence. Five of the HCRs tested decreased mRNA steady-state levels 2- to 10-fold relative to controls, presumably by altering mRNA stability. One HCR increased translation, and one decreased translation. Elevated mitogen levels caused four HCRs to increase protein levels twofold. One HCR increased protein levels fourfold in response to hypoxia. Although nonconserved UTR sequences may also have a role, these results provide evidence that sequences that are highly conserved during evolution are good candidates for RNA motifs with posttranscriptional regulatory functions in gene expression.

Cooperative interaction of branch signals in the actin intron of Saccharomyces cerevisiae.

In pre-mRNA splicing, specific spliceosomal components recognize key intron sequences, but the mechanisms by which splice sites are selected arenot completely understood. In the Saccharomyces cerevisiae actin intron a silent branch point-like sequence (UACUAAG) is located 7 nt upstream of the canonical sequence. Mutation of the canonicalUACUAAC sequence to UAAUAAC reduces utilization of this signal and activates the cryptic UACUAAG. Splicing-dependent beta-galactosidase assays have shown that these two splice signals cooperate to enhance splicing. Analyses of several variants of this double branch point intron demonstrate that the upstream UACUAAG sequence significantly increases usage of the UAAUAAC as a site of lariat formation. This activation is sequence-specific and unidirectional. However the ability of the UACUAAG signal to activate the downstream branch point is dependent on the presence of a short non-conserved sequence located a few nucleotides upstream of the UACUAAG. Mutation of this sequence leads to the disappearance of the cooperative interactions between the two branch signals. Our results show that this non-conserved sequence and the UACUAAG signal must both be present to achieve activation of the downstream branch point and suggest that a specific structure may be necessary to allow efficient recognition of the UAAUAAC.

Histone Synthesis inTrypanosoma cruzi

Trypanosoma cruziis an ancient, parasitic eukaryote which does not undergo chromatin condensation during cell division. This behavior may be explained if one considers the strong amino acid sequence divergence ofTrypanosomahistones compared to higher eukaryotes. In the latter organisms histone synthesis is coupled to DNA replication. Considering the nonconserved amino acid sequence ofT. cruzihistones, as well as the absence of chromatin condensation in this organism, we have studied histone synthesis in relation to DNA replication in this parasite. We have found that core histones and a fraction of histone H1 are synthesized concomitantly to DNA replication. However, another fraction of histone H1 is constitutively synthesized.

Identification and classification of Oxalobacter formigenes strains by using oligonucleotide probes and primers.

Genomic DNAs of various strains of Oxalobacter formigenes were subjected to restriction endonuclease fragment length polymorphism- and PCR-based amplification analyses with DNA probes and primers complementary to sequences within either the oxc gene, encoding oxalyl coenzyme A (oxalyl-CoA) decarboxylase, or the frc gene, encoding formyl-CoA transferase. Oligonucleotide probes based on nonconserved sequences of oxc or frc were able to divide O. formigenes strains into at least two groups, consistent with the current separation of O. formigenes strains into groups I and II on the basis of 16S rRNA sequence similarities and lipid content. In contrast, an oligonucleotide probe based on the conserved 5' end of oxc appeared to bind all group I and the majority of group II strains. PCR amplification of the oxc gene showed even greater sensitivity in detecting O. formigenes and provided support for further division of the strains into subgroups. In addition, these oligonucleotides failed to hybridize to or amplify PCR products from whole fecal DNA isolated from fresh stool samples from an individual not colonized with O. formigenes, indicating unique specificity. Thus, these DNA analyses permit both detection as well as classification of O. formigenes strains.

Monoclonal antibodies specific for Semliki Forest virus replicase protein nsP2.

A panel of monoclonal antibodies (MAbs) was raised against Semliki Forest virus (SFV) nonstructural protein nsP2, which is a protease, an NTPase, a putative RNA helicase, and a regulator of the synthesis of the subgenomic 26S mRNA encoding the structural proteins. nsP2, used for immunization, was expressed as a histidine fusion protein in Escherichia coli and purified by metal affinity chromatography. Dot-blot assay, using a membrane fraction from SFV-infected cells as antigen, gave 33 positive clones. Of these, 30 MAbs recognized nsP2 in Western immunoblotting, and 25 showed positive indirect immunofluorescence (IFAT) in SFV-infected cells; 15 MAbs stained the cytoplasmic vacuoles (CPVI), which are the sites of viral RNA synthesis in alphavirus-infected cells. MAb 3B5 recognized only CPVIs, as shown by double immunofluorescence staining with polyclonal anti-nsP3 antiserum. Most of the MAbs (20/33) recognized the nuclear form of nsP2, which may be associated with SFV neurovirulence. Immunoprecipitation with MAbs revealed that the SFV nonstructural proteins are associated with each other. None of the MAbs recognized Sindbis virus nsP2 in immunoblotting, indicating that they were directed to non-conserved sequences specific for SFV. Interestingly, these epitopes were located mostly within the N-terminal half of nsP2. Unexpectedly, the anti-nsP2 MAb 1E9 cross-reacted strongly with a host protein of 78 kDa from uninfected human, murine, avian and insect cells. This protein was identified as the immunoglobulin binding protein, BiP, by 2-D gel mapping and reaction with anti-BiP antiserum.

Mutations in the nonconserved noncoding sequences of the influenza A virus segments affect viral vRNA formation

Influenza A virus replication and packaging is mediated by cis-acting signals, which are located at the 3′ and the 5′ end of the viral segments. The terminal residues can be divided into conserved and nonconserved residues. We have constructed a mutant influenza A/WSN/33 virus, which contains multiple mutations in the nonconserved residues of the neuraminidase (NA) segment. This virus shows a segment-specific reduction of the genomic RNA content in the infected cell and in the progeny virus. Further mutants and revertant viruses revealed that it was not possible to define specific residues, which were responsible for the reduction of the NA-specific RNA. Thus, it appears that an efficient vRNA formation is dependent on the synergistic effect of the terminal sequences.

Challenges of three-dimensional reconstruction of ribonucleoprotein complexes from electron spectroscopic images - Reconstructing ribosomal RNA

Transmission electron microscopy has been dominant in structural studies of the ribosome and its constituent ribonucleic acids and proteins. Ribosomal RNA (rRNA) has central importance in the architecture of this complex and in protein synthesis. Our work has entailed using electron spectroscopic imaging (ESI) to probe the tertiary structure of rRNAin situin a prokaryote (Escherichia coli) and in a eukaryote (Thermomyces lanuginosus). ESI uses only electrons which have lost a specific amount of energy due to specific inner-shell ionisation interactions with the specimen to form an elemental map. In nucleoprotein complexes, a map of the phosphorus distribution represents primarily a projection of the phosphate backbone of the nucleic acid component. The visualisation of rRNAin situin the intact ribosomal subunit by ESI was demonstrated almost a decade ago to be feasible. Our work on quantitative image analysis of ES images ofE. coliandTh. lanuginosusribosomal subunits has presented unique challenges and has resulted in new algorithmic developments generally applicable to such images. These innovations include a singular pretreatment procedure, the use of mutual correlation functions rather than cross correlation functions to reduce the effect of low spatial frequency components, and angular determination using iterative quaternion-assisted angular reconstitution to compute a three-dimensional reconstruction. These investigations have produced direct information regarding ribosomal rRNA localisation in the ribosomal subunits ofE. coliandTh. lanuginosus, and the position of non-conserved sequences.

Multiple DNA elements in ARS305 determine replication origin activity in a yeast chromosome.

A yeast autonomously replicating sequence, ARS305, shares essential components with a chromosome III replicator, ORI305. Known components include an ARS consensus sequence (ACS) element, presumed to bind the origin recognition complex (ORC), and a broad 3'-flanking sequence which contains a DNA unwinding element. Here linker substitution mutagenesis of ARS305 and analysis of plasmid mitotic stability identified three short sequence elements within the broad 3'-flanking sequence. The major functional element resides directly 3' of the ACS and the two remaining elements reside further downstream, all within non-conserved ARS sequences. To determine the contribution of the elements to replication origin function in the chromosome, selected linker mutations were transplaced into the ORI305 locus and two-dimensional gel electrophoresis was used to analyze replication bubble formation and fork directions. Mutation of the major functional element identified in the plasmid mitotic stability assay inactivated replication origin function in the chromosome. Mutation of each of the two remaining elements diminished both plasmid ARS and chromosomal origin activities to similar levels. Thus multiple DNA elements identified in the plasmid ARS are determinants of replication origin function in the natural context of the chromosome. Comparison with two other genetically defined chromosomal replicators reveals a conservation of functional elements known to bind ORC, but no two replicators are identical in the arrangement of elements downstream of ORC binding elements or in the extent of functional sequences adjacent to the ACS.

An alanine racemase gene as a new index for detecting Escherichia coli in foods.

A gene of alanine racemase, a typical prokaryotic enzyme, was evaluated as a new index for detecting Escherichia coli in foods. An alanine racemase gene fragment containing a non-conserved sequence of the gene was amplified from genomic DNA of E. coli by a polymerase chain reaction, and then labeled with digoxigenin as a probe for detecting E. coli. Food samples and bacteria were each treated as at 25 degrees C for 10 min in 0.1N NaOH containing 0.5% SDS, before being directly spotted on to nylon membranes for DNA hybridization. The probe was specific for E. coli; all 48 strains of E. coli examined, including such pathogenic strains as E. coli O157:H7, showed positive signals, whereas all 59 strains of non-E. coli species, except for one strain (Shigella sonnei), did not show a signal. Various foods inoculated with E. coli K-12 showed positive signals whereas no uninoculated foods showed any signal. Quantification of E. coli cells in the death phase by the hybridization method showed good correlation with that by the plate culture method. The alanine racemase gene could prove useful as an index for detecting E. coli in foods.

Evolutionary conservation in the DNA-binding and -bending properties of HMG-boxes from SRY proteins of primates

Mammalian sex determination is caused by the Y-chromosome gene SRY, which encodes a protein containing a DNA-binding domain (HMG-box) of about 70 amino acids (aa). The HMG-box is very conserved in a wide variety of mammals; conversely, the flanking non-box regions show a high degree of aa sequence divergence, even between closely related species. The HMG-box of human SRY binds sequence-specifically to linear DNA and produces a sharp bend; it also interacts with high affinity to kinked DNA structures irrespective of their sequences. Point mutations associated with sex reversal in XY human females fall within the HMG-box and either affect the affinity for DNA or modify the geometry of the DNA-protein complex. Here, we show that the DNA-binding and -bending properties of the HMG-boxes of SRY from human and seven different primates are extremely similar to each other. Together with other data, this suggests that the inability of mouse and human SRY to substitute for each other is due to differences in the conserved HMG-box, rather than the non-conserved flanking sequences.

Induction of antibodies to the Plasmodium falciparum merozoite surface protein-1 (MSP1) by cross-priming with heterologous MSP1s.

The merozoite surface protein-1 (MSP1) of Plasmodium falciparum possesses intervening conserved and nonconserved sequences. The relative importance of these sequences in providing T cell help for Ab production was investigated in a series of cross-priming studies using homologous and heterologous parasite MSP1 proteins. Cross-priming with heterologous MSP1s was as efficient as homologous immunizations in inducing anti-MSP1 Abs. Similar to homologous immunization, cross-priming with heterologous MSP1s induced primarily Abs to conserved epitopes. The specificities of the Abs were also similar for the two immunization regimens. Studies were also performed with use of the C-terminal p42 fragment of MSP1 expressed in baculovirus (BVp42). When BVp42 was used either as the priming Ag followed by boosting with homologous (or heterologous) MSP1 or as the booster Ag after priming with homologous (or heterologous) MSP1, much lower anti-BVp42 Ab titers were produced compared with priming/boosting with homologous or heterologous MSP1s or BVp42 alone. Thus, immunization with the complete parasite MSP1 induced a dominant, conserved Th epitope(s) specific for anti-p42 Ab production, and such determinant(s) was either located outside the p42 region or was not provided by the BVp42 because of possible differences in the processing of parasite MSP1 vs BVp42. Our data provided a strong rationale to identify and include conserved Th epitope(s) in MSP1 vaccines. Furthermore, a MSP1 vaccine on the basis of the C-terminal p42 fragment may benefit by the inclusion of additional Th epitopes to achieve effective boosting in the field.

The binding site of the transcriptional activator VirG from Agrobacterium comprises both conserved and specific nonconserved sequences

Virulence genes of Agrobacterium tumefaciens are transcriptionally activated in response to phenolic compounds and certain sugars. The transcriptional activator VirG specifically binds to fragments containing the conserved vir box sequence present in the promoter region of all vir genes. This study shows that both the vir box as well as specific nonconserved sequences downstream of the vir box are required for VirG binding and transcriptional activation. Insertion of the identified VirG binding site into the lac promoter resulted in transcriptional activation of this heterologous promoter in response to the plant phenolic signal molecule acetosyringone.

Antibodies against highly conserved sites in the epidermal growth factor receptor tyrosine kinase domain as probes for structure and function.

We generated anti-peptide antibodies against four highly conserved sequences in the kinase domain and against two nonconserved sequences surrounding autophosphorylation sites in the carboxyl-terminal domain of the epidermal growth factor receptor (EGFR). These antibodies were used to examine topology and function in catalysis of specific sequences. Two of the highly conserved sites, HRD (residues 811-818) and DFG (residues 827-838), appeared to participate in catalysis since alpha HRD and alpha DFG but not the other anti-peptide antibodies inhibited EGFR kinase activity. Examination of the topology of the six sites revealed that epitopes in all except the HRD site appeared to be exposed to antibody binding in the EGFR. The conditions that caused increased exposure of the HRD site to interaction with antibody included autophosphorylation, addition of the ionic detergent sodium dodecyl sulfate (SDS), and elevation in temperature from 4 to 34 degrees C.

Functional properties of pro region of Escherichia coli heat-stable enterotoxin.

Escherichia coli heat-stable enterotoxin Ip (STp) is synthesized as the 72-amino-acid residue precursor consisting of three regions: pre region (amino acid residues 1 to 19), pro region (amino acid residues 20 to 54), and mature ST (mST) region (amino acid residues 55 to 72). We examined the role of the pro sequence of STp in enterotoxigenicity of a strain by deleting the gene fragment encoding amino acids 22 to 57. This deletion caused a remarkable reduction of its enterotoxic activity of culture supernatant. In order to analyze the sequence responsible for the function of the pro region, two additional deletion mutants were made. The deletion of the sequence covering amino acids 29 to 38, which is conserved in all sequences of ST reported, brought about a significant reduction of enterotoxic activity but the deletion of the non-conserved sequence (amino acids 40 to 53) did not. This result shows that conserved sequence is mainly responsible for the function. Subsequently, to examine the mechanism of action of the pro region, plasmids carrying DNA sequences of hybrid proteins consisting of pre-pro-nuclease, pre-mST-nuclease, pre-pro-mST-nuclease and pre-pro-nuclease-mST were constructed. Amino acid sequence determination and SDS-polyacrylamide gel analysis revealed that these fusion proteins were cleaved between pre sequence and pro sequence during secretion and the cleaved fusion proteins were accumulated in periplasmic space. But the amount of hybrid protein accumulated in the periplasmic space varied among the strains. That is, the amount of the pre-pro-nuclease gene product that accumulated in the periplasmic space was the highest of all fusion gene products. These results indicate that the existence of the mST region strongly interferes with the translocation of the gene product into the periplasmic space and that the pro region functions to guide the mST region into the periplasmic space.

Mutational analysis of the adenovirus 2 IVa2 initiator and downstream elements.

The initiator element of the adenovirus type 2 IVa2 promoter is sufficient to direct accurate initiation by RNA polymerase II. Analysis of the effects of substitution of specific base pairs on initiator activity in in vitro transcription systems indicated that specific sequences between positions -4 and +5 were essential for initiator activity. Mutations that impaired or eliminated initiator activity altered both base pairs that are conserved in sequence-related initiators and nonconserved sequences. Neither the downstream TA-rich sequence of the IVa2 promoter, nor the adenovirus 2 major late TATA element placed at the same downstream site could overcome the severe inhibitory effects of initiator mutations, indicating that the initiator is the primary determinant of the specificity and direction of IVa2 transcription. By contrast, when the ML TATA element was placed 31 nucleotides upstream of the IVa2 initiator, the precise specificity, but neither the efficiency nor direction of transcription, depended on the presence of a functional initiator. Activity of the IVa2 promoter was relatively insensitive to changes in the orientation or nature of the TA-rich sequence. Furthermore, only a promoter containing the ML TA-TAAAA sequence downstream of the IVa2 initiator was competent to direct both IVa2 transcription and transcription from the opposite strand. The implications of this functional difference for recognition of the downstream element are discussed.

Structural and functional analysis of the amdR regulatory gene of Aspergillus oryzae

We have isolated the Aspergillus oryzae homologue of the amdR regulatory gene of Aspergillus nidulans by cross hybridization. Sequence analysis and functional studies have shown that the amdR genes are highly conserved and functionally interchangeable between the two species. The homology between the two genes extends throughout most of the coding sequences, including sequences encoding the DNA-binding domain and putative activation domains. Two regions of nonconserved sequence were also identified. Studies using various amdS:: lacZ fusion constructs indicate that the A. oryzae gene product binds similar sequences and responds to inducer in a similar manner to the A. nidulans protein. Inactivation of the A. oryzae gene results in the inability to grow on y-amino-butyric acid (GABA) as a carbon and/or nitrogen source indicating that GABA utilization is amdR-dependent in A. oryzae as it is in A. nidulans.

Nucleotide sequence of the capsid protein gene of two serotypes of San Miguel sea lion virus: Identification of conserved and non-conserved amino acid sequences among calicivirus capsid proteins

The San Miguel sea lion viruses, members of the calicivirus family, are closely related to the vesicular disease of swine viruses which can cause severe disease in swine. In order to begin the molecular characterization of these viruses, the nucleotide sequence of the capsid protein gene of two San Miguel sea lion viruses (SMSV), serotypes 1 and 4, was determined. The coding sequences for the capsid precursor protein were located within the 3' terminal 2620 bases of the genomic RNAs of both viruses. The encoded capsid precursor proteins were 79,500 and 77,634 Da for SMSV 1 and SMSV 4, respectively. The SMSV 1 protein was 47.7% and SMSV 4 was 48.6% homologous to the feline calicivirus (FCV) capsid precursor protein while the two SMSV capsid precursors were 73% homologous to each other. Six distinct regions within the capsid precursors (denoted as regions A–F) were identified based on amino acid sequence alignment analysis of the two SMSV serotypes with FCV and the rabbit hemorrhagic disease virus (RHDV) capsid protein. Three regions showed similarity among all four viruses (regions B, D and F) and one region showed a very high degree of homology between the SMSV serotypes but only limited similarity with FCV (region A). RHDV contained only a truncated region A. A fifth region, consisting of approximately 100 residues, was not conserved among any of the viruses (region E) and, in SMSV, may contain the serotype-specific determinants. Another small region (region C) contained between 15 and 27 amino acids and showed little sequence conservation. Region B showed the highest degree of conservation among the four viruses and contained the residues which had homology to the picornavirus VP3 structural protein. An open reading frame, found in the 3' terminal 514 bases of the SMSV genomes, encoded small proteins (12,575 and 12,522 Da, respectively for SMSV 1 and SMSV 4) of which 32% of the conserved amino acids were basic residues, implying a possible nucleic acid-binding function.

Identification of hepatitis C viruses with a nonconserved sequence of the 5' untranslated region.

The nucleotide sequence of the 5' untranslated region of hepatitis C virus (HCV) has been shown to be conserved. In contrast, we have detected more sequence variation in this region in several HCV isolates than hitherto expected. The nucleotide sequences of the 5' untranslated regions of these isolates differ significantly from that of the prototype but are very similar to each other. We believe that these isolates belong to the same type of HCV. Among 240 HCV RNA polymerase chain reaction-positive specimens that we examined, 7 belong to this type. The results suggest that the HCV variants that we detected represent a different type of HCV and that they are responsible for approximately 3% of HCV infections in patients that we have examined.