Sequence S1 Research Articles

Complete nucleotide sequence of Colorado tick fever virus segments M6, S1 and S2.

The nucleotide sequences of the tenth (M6), eleventh (S1) and twelfth (S2) dsRNA genomic segments of the Florio strain (N-7180) of Colorado tick fever virus were determined and found to be 675, 998 and 1884 bp, respectively, in length. A nonanucleotide motif and a hexanucleotide motif were found to be highly conserved in the 5' and 3' non-coding regions (NCRs), respectively, of the three segments. The first and last three nucleotides of each segment were of inverted complementarity, and segment-specific inverted terminal repeats were detected in the NCRs of the three segments. These findings suggest the occurrence of intracellular panhandle structures for the RNA transcripts. A readthrough phenomenon is suspected in segment M6. The environment surrounding the opal codon (position 1052-1054) of segment M6 conforms to that of leaky opal codons described in the literature.

Involvement of S2 episomal sequences in the generation of NCS4 deletion mutation in maize mitochondria.

Cytoplasmic male sterility (CMS) and the abnormal-growth, nochromosomal stripe (NCS) phenotypes are cytoplasmically determined traits in plants. Mitochondrial DNA rearrangements involving short repeats appear to be responsible for the production of CMS reversions to fertility and NCS mutations. NCS4, a new mutant of maize CMS-S, exhibits both abnormal growth and male fertility. This mutant is unique because both mutations occurred within the same plant. Free S1 and S2 episomes normally found in CMS-S mitochondria have been lost from NCS4 plants. An S2 sequence has recombined aberrantly with a ribosomal protein coding region, rps3/rpl16. One end of the S2 sequence and the 5' end of the rps3/rpl16 transcription unit are absent from the NCS4 recombinant genome. Loss of mitochondrial ribosomal protein function is lethal; therefore, NCS4 plants are heteroplasmic for the rps3 deletion. Loss of S sequences from CMS-S mitochondria is not lethal and plants regain pollen function. Thus, although NCS4 plants have very abnormal plant phenotypes, they are male-fertile.

Coassembly of synthetic segments of shaker K+ channel within phospholipid membranes.

Increasing evidence suggests that membrane-embedded hydrophobic segments can interact within the phospholipid milieu of the membrane with varying degrees of specificity and thus contribute to the folding and oligomerization of proteins. We have used synthetic peptides corresponding to segments from the hydrophobic core of the Shaker potassium channel as a model system to study interactions between membrane-embedded segments. Three synthetic segments of the Shaker K+ channel, comprising the hydrophobic S2, S3, and S4 sequences, were used, and their secondary structure, their interactions with, and orientation within phospholipid membranes were examined. Secondary structure studies revealed that though S3 and S4 both adopt certain fractions of alpha-helical structures in membrane mimetic environments, the alpha-helical content of S3 is lower. Both S3 and S4 bind strongly to zwitterionic phospholipids, with partition coefficients in the order of 10(4) and 10(5) M-1. ATR-FTIR studies showed that while the S4 peptide is oriented parallel to the membrane surface, S3 tends to a more transmembranal orientation. Enzymatic cleavage experiments demonstrated that the presence of S3 induces some change in the proteolytic accessibility of the S4 segment. Resonance energy transfer measurements, done in high lipid/peptide molar ratios, revealed that S3 and S4 cannot self-associate in zwitterionic phospholipid vesicles but can associate with each other and with the S2 segment of the channel. Furthermore, S3 does not interact with the homologous S4 region from the first repeat of the eel sodium channel, demonstrating specificity in the interactions. These results are in line with data indicating that functionally important interactions indeed exist between the negatively charged S2 and S3 regions and the positively charged S4 region [Papazian, D. M., et al (1995) Neuron 14, 1293-1301; Planells-Cases, R., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 92, 9422-9426]. From a broader point of view, these results provide further support to the notion that interactions (either specific or nonspecific) may exist between transmembrane segments of integral membrane proteins and therefore can contribute to their assembly and organization.

Distribution and action of SALMFamide neuropeptides in the starfish Asterias rubens.

The SALMFamides S1 and S2 are two structurally related neuropeptides that are present in starfish, and which share the C-terminal amino acid sequence SXLXFamide, where X is variable. To establish the distribution of S1 and S2 in starfish, we have raised antisera that recognise specifically the C-terminal pentapeptide sequence of either S1 or S2. Here we describe the production and characterisation of an S2-specific antiserum designated CLII. This antiserum, together with an S1-specific antiserum (BLII), has been used in a radioimmunoassay to measure S1 and S2 levels in extracts of body parts from the starfish Asterias rubens. High concentrations (250-400 pmol g-1) of both peptides were detected in the radial nerve cords of the nervous system and lower concentrations were detected in other body parts, including neuromuscular organs such as the tube feet, apical muscle and cardiac stomach. We have examined the pharmacological effects of S1 and S2 on the contractility of these three preparations. Neither S1 nor S2 influenced the tone of tube foot and apical muscle preparations but S2 caused relaxation of cardiac stomach preparations, antagonising the contracting action of acetylcholine.

Characterization of transposon Tn5469 from the cyanobacterium Fremyella diplosiphon

A transposon, designated Tn5469, was isolated from mutant strain FdR1 of the filamentous cyanobacterium Fremyella diplosiphon following its insertion into the rcaC gene. Tn5469 is a 4,904-bp noncomposite transposon with 25-bp near-perfect terminal inverted repeats and has three tandemly arranged, slightly overlapping potential open reading frames (ORFs) encoding proteins of 104.6 kDa (909 residues), 42.5 kDa (375 residues), and 31.9 kDa (272 residues). Insertion of Tn5469 into the rcaC gene in strain FdR1 generated a duplicate 5-bp target sequence. On the basis of amino acid sequence identifies, the largest ORF, designated tnpA, is predicted to encode a composite transposase protein. A 230-residue domain near the amino terminus of the TnpA protein has 15.4% amino acid sequence identity with a corresponding domain for the putative transposase encoded by Lactococcus lactis insertion sequence S1 (ISS1). In addition, the sequence for the carboxyl-terminal 600 residues of the TnpA protein is 20.0% identical to that for the TniA transposase encoded by Tn5090 on Klebsiella aerogenes plasmid R751. The TnpA and TniA proteins contain the D,D(35)E motif characteristic of a recently defined superfamily consisting of bacterial transposases and integrase proteins of eukaryotic retroelements and retrotransposons. The two remaining ORFs on Tn5469 encode proteins of unknown function. Southern blot analysis showed that wild-type F. diplosiphon harbors five genomic copies of Tn5469. In comparison, mutant strain FdR1 harbors an extra genomic copy of Tn5469 which was localized to the inactivated rcaC gene. Among five morphologically distinct cyanobacterial strains examined, none was found to contain genomic sequences homologous to Tn5469.

Extended skolem sequences

AbstractA k‐extended Skolem sequence of order n is an integer sequence (s1, s2,…, s2n+1) in which sk = 0 and for each j ϵ {1,…,n}, there exists a unique i ϵ {1,…, 2n} such that si = si+j = j. We show that such a sequence exists if and only if either 1) k is odd and n ≡ 0 or 1 (mod 4) or (2) k is even and n ≡ 2 or 3 (mod 4). The same conditions are also shown to be necessary and sufficient for the existence of excess Skolem sequences. Finally, we use extended Skolem sequences to construct maximal cyclic partial triple systems. © 1995 John Wiley & Sons, Inc.

Maize S1 and S2 sequences in wheat mitochondrial DNA

The mitochondrial DNA (mtDNA) of fertile ( Triticum aestivum) and cytoplasmic male sterile (cms) wheat ( T. aestivum nucleus; T. timopheevi mitochondria) was isolated and subjected to restriction enzyme analysis. Restriction fragments of the mtDNAs were probed with DNA specific to the S-episomes of maize mitochondria. Small, independent episomal DNA was not found in the mtDNA of wheat. Sequences related to maize S1 DNA are present in fertile and cms wheat mtDNA but do not appear to be contiguous. Restriction enzyme analyses do not show detectable differences between fertile and cms mtDNA around the maize-like S1 sequences. Sequences related to maize S2 mtDNA may be located at two different positions in both fertile and cms mitochondrial genomes. The fertile and cms mtDNA show restriction fragment length polymorphism at one of two sites.

EXTENDED ELECTRONIC STATES IN A CLASS OF ONE DIMENSIONAL QUASIPERIODIC SYSTEMS

Using the dynamical-map method, we study the electronic properties for diagonal and off-diagonal tight-binding model in a class of one-dimensional quasiper-iodic systems. These systems are associated with the generalized Fibonacci sequences S∞ given by the recursion relation Sl+1=Sl2j-1Sl-12i for l≥1 with two initial sequences S0 and S1, where i and j are positive integers. Analytical treatments show that the extended electronic states for these one-dimensional quasiperiodic sequences really exist.

The S2 gene nucleotide sequences of prototype strains of the three reovirus serotypes: characterization of reovirus core protein sigma 2

The S2 gene nucleotide sequences of prototype strains of the three reovirus serotypes were determined to gain insight into the structure and function of the S2 translation product, virion core protein sigma 2. The S2 sequences of the type 1 Lang, type 2 Jones, and type 3 Dearing strains are 1,331 nucleotides in length and contain a single large open reading frame that could encode a protein of 418 amino acids, corresponding to sigma 2. The deduced sigma 2 amino acid sequences of these strains are very conserved, being identical at 94% of the sequence positions. Predictions of sigma 2 secondary structure and hydrophobicity suggest that the protein has a two-domain structure. A larger domain is suggested to be formed from the amino-terminal three-fourths of sigma 2 sequence, which is separated from a smaller carboxy-terminal domain by a turn-rich hinge region. The carboxy-terminal domain includes sequences that are more hydrophilic than those in the rest of the protein and contains sequences which are predicted to form an alpha-helix. A region of striking similarity was found between amino acids 354 and 374 of sigma 2 and amino acids 1008 and 1031 of the beta subunit of the Escherichia coli DNA-dependent RNA polymerase. We suggest that the regions with similar sequence in sigma 2 and the beta subunit form amphipathic alpha-helices which may play a related role in the function of each protein. We have also performed experiments to further characterize the double-stranded RNA-binding activity of sigma 2 and found that the capacity to bind double-stranded RNA is a property of the sigma 2 protein of prototype strains and of the S2 mutant tsC447.

Structure and expression of the human gene encoding testicular H1 histone (H1t)

The gene coding for the human H 1t histone, a testis-specific H1 subtype, was isolated from a genomic library using a human somatic H1 gene as a hybridization probe. The corresponding mRNA is not polyadenylated and encodes a 206-amino-acid protein. Sequence analysis and S1 nuclease mapping of the human H1t gene reveals that the 5' flanking region contains several consensus promoter elements, as described for somatic, i.e., S-phase-dependent H1 subtype genes. The 3' region includes the stem-and-loop structure necessary for mRNA processing of most histone mRNAs. Northern blot analysis with RNAs from different human tissues and cell lines revealed that only testicular RNA hybridized with this gene probe.

Isolation of preferentially expressed genes in the kidneys of hypertensive rats.

By differential hybridization, three complementary DNAs designated as S3, S2, and SA were isolated, and the corresponding messenger RNAs (mRNAs) were differentially expressed between the kidneys of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. S3 is identical to cytochrome P450 IV A2. SA encoded a protein of 546 amino acid residues, and its carboxyl terminal region had a slight homology to luciferase. No homologous sequence has been reported in S2 sequences. S3 mRNA was about four times more abundantly expressed in the kidneys of 28-day-old SHR than in those of age-matched WKY rats, but there was no difference at age 16 weeks. A low NaCl diet positively modulated the expression of the S3 gene. S2 mRNA was almost undetectable in the kidneys of 28-day-old WKY rats but was clearly detected in those of age-matched SHR. The expression level of S2 mRNA in the livers of 16-week-old SHR was about five times higher than that of age-matched WKY rats. The expression of S2 mRNA in the livers was modulated by dietary NaCl and captopril. SA mRNA was more than 10 times more abundantly expressed in the kidneys of SHR than in those of WKY rats from age 4 weeks. With the administration of captopril, the expressions of SA mRNA in the livers of SHR were positively modulated. Because these three genes are not only differentially expressed between SHR and WKY rats but also related to sodium metabolism or blood pressure control, the identification of these genes may provide important probes to examine the mechanisms of hypertension.

Sequence and expression of the Escherichia coli recR locus

The Escherichia coli RecR protein participates in a recombinational DNA repair process. Its gene is located in a region of chromosome that extends from 502 to 509 kilobases on the physical map and that contains apt, dnaX, orf12-recR, htpG, and adk. Most, if not all, of these are involved in nucleic acid metabolism. The orf12-recR reading frames consist of 935 base pairs and overlap by one nucleotide, with the 3' A of the orf12 termination codon forming the 5' nucleotide of the recR initiation codon. The orf12-recR promoter was located upstream of orf12 by sequence analysis, promoter cloning, and S1 nuclease protection analysis. The start point of transcription was determined by primer extension. The transcript 5' end contained a long, apparently untranslated region of 199 nucleotides. Absence of a detectable promoter specific for recR and the overlap of the orf12 and recR reading frames suggest that translation of recR is coupled to that of orf12. By maxicell analysis, it was determined that both orf12 and recR are translated.

Recognition of pertussis toxin by antibodies to synthetic peptides

Eight synthetic peptides, selected from the amino acid sequence of pertussis toxin (PT) subunits S1, S2, S3 and S4, were assessed for their ability to induce protein-recognizing and neutralizing antibodies. Seven of these peptides, prepared as conjugates of either keyhole limpet haemocyanin or tetanus toxoid, induced significant levels of antibody, all of which reacted with SDS-denatured PT on Western blots. Six of the antibodies bound to PT-coated ELISA plates; this binding was inhibited by homologous peptide antigen. However, none of the antibodies, including those directed against the N-eterminus of subunit S1, were able to attenuate in vivo or in vitro toxin-dependent activity. Further investigation revealed that only one antibody, specific for the C-terminus of S1 (peptide Slc, 237–255), could recognize the conformationw of native PT in solution. The other five antipeptide antibodies which reacted with PT-coated ELISA plates did not recognize PT when captured onto ELISA plates via either a monoclonal antibody or fetuin, unless the conformation of the toxin had been relaxed by reduction with dithiothreitol. Conversely, the native PT-recognizing response of peptide Slc did not bind the conformationally relaxed PT molecule. From this study, it appears likely that a peptide capable of inducing PT-neutralizing antibody must closely resemble the conformation of the cognate sequence in the native protein.

Positive and negative control of translation by the leader sequence of cauliflower mosaic virus pregenomic 35S RNA.

We have studied the influence of the 600 nt long leader sequence of cauliflower mosaic virus 35S RNA on downstream translation. Plant protoplasts were transfected with plasmids expressing a CAT reporter gene from a mRNA, containing wild-type or mutant forms of the 35S RNA leader. Deletion analysis revealed the presence of three separate stimulatory sequence regions, S1, S2 and S3. The latter two interact with each other to enhance downstream translation 5- to 10-fold. This enhancement was not observed in protoplasts from a non-host plant. In the absence of either S2 or S3, the region I2, located in between, exerts an inhibitory effect on downstream translation, probably due to the presence of short open reading frames. Expression of a reporter gene inserted into I2 increases 2-fold upon deletion of either S2 or S3. We propose that mRNA regions S2 and S3 form a complex with cellular factors that allows scanning ribosomes to bypass region I2.

Differential expression of two related organ-specific genes in pea

We have screened a pea genomic library using a cDNA probe derived from pea shoot RNA. From this screen, we isolated two closely related genes, designated as S2 and P4. An intriguing property of these two genes is the presence in their coding region of a repeated sequence that is conserved between them in sequence but not in the number of the repeating units. The predicted amino acid sequence suggests that these proteins could be exported and glycosylated. 3' S1 analysis reveals that one of the genes, S2, is expressed highly in stem, as expected from previous work. However, mRNA derived from the other gene, P4, is not detectable in stem tissue, but is present in tissue derived from pea pods. The 5' upstream sequence of S2 and P4 are 94% identical up to position -121, suggesting that sequences upstream of -121 are responsible for organ-specific expression of the two genes.

Periodic and quasiperiodic wavefunctions in a class of one-dimensional quasicrystals: an analytical treatment

The one-dimensional, discrete Schrodinger equation is studied analytically for a class of quasiperiodic Hamiltonians known as the copper mean. The lattice is described by a recursion formula SL+1=SL-1SL-1SL, L=2,3, ., given the two initial sequences S0 and S1. Extended states are shown to exist for energies satisfying TrTL=0 and Tr((T0)-2T1)=2 where TL is the transfer matrix for the Lth generation of the quasicrystal. Also, periodic states are shown to exist quite generally in a subclass of the copper mean. A specific one-dimensional quasicrystal is given as an example of this, and is shown to have exclusively periodic states.

Expression from the transferrin gene promoter in transgenic mice.

Transferrin is an iron-binding protein that is expressed as a major product in liver and secreted into the plasma. To study the tissue-specific regulatory regions of this gene, the genomic mouse transferrin (mTf) gene was cloned and characterized by partial sequence analysis and S1 nuclease mapping of the transcriptional start site. Fusion genes containing the transferrin gene promoter and 5'-flanking sequences were ligated to the human growth hormone (hGH) gene and used to produce transgenic mice. A deletion construct containing the -581 to +50 region of the transferrin gene was sufficient to direct a high level of liver-specific expression resembling endogenous transferrin gene expression. Deletion to -139 base pairs of 5'-flanking sequence gave a construct which retained liver specificity, but the magnitude of expression decreased severalfold. These results demonstrate the presence of a liver-specific transcriptional element between -139 and +50 and suggest the presence of a distal element between -581 and -139 that can further increase expression. Surprisingly, fusion constructs containing -3 kilobase pairs (kb) of 5'-flanking sequence gave higher levels of mRNA in nonhepatic tissues than did either the -581 or -139 construct. Further studies indicated that the high levels of circulating hGH in these transgenic mice specifically induced the endogenous transferrin and albumin genes in liver and also stimulated the normally low levels of expression of the endogenous transferrin gene in brain, heart, kidney, and muscle. A mutated hGH gene that does not produce active growth hormone was fused to the -3- to +50-kb transferrin sequences to produce the -3-kb mTf-hGX construct. A liver-specific pattern of expression was observed in transgenic mice harboring the -3-kb mTf-hGX construct, and this mutated transgene was shown to be induced four- to sevenfold by either bovine or human growth hormone. These results demonstrate the presence of a growth hormone-responsive element between -3 and +50 kb in the 5'-flanking region of the mTf gene promoter.

Expression from the transferrin gene promoter in transgenic mice.

Transferrin is an iron-binding protein that is expressed as a major product in liver and secreted into the plasma. To study the tissue-specific regulatory regions of this gene, the genomic mouse transferrin (mTf) gene was cloned and characterized by partial sequence analysis and S1 nuclease mapping of the transcriptional start site. Fusion genes containing the transferrin gene promoter and 5'-flanking sequences were ligated to the human growth hormone (hGH) gene and used to produce transgenic mice. A deletion construct containing the -581 to +50 region of the transferrin gene was sufficient to direct a high level of liver-specific expression resembling endogenous transferrin gene expression. Deletion to -139 base pairs of 5'-flanking sequence gave a construct which retained liver specificity, but the magnitude of expression decreased severalfold. These results demonstrate the presence of a liver-specific transcriptional element between -139 and +50 and suggest the presence of a distal element between -581 and -139 that can further increase expression. Surprisingly, fusion constructs containing -3 kilobase pairs (kb) of 5'-flanking sequence gave higher levels of mRNA in nonhepatic tissues than did either the -581 or -139 construct. Further studies indicated that the high levels of circulating hGH in these transgenic mice specifically induced the endogenous transferrin and albumin genes in liver and also stimulated the normally low levels of expression of the endogenous transferrin gene in brain, heart, kidney, and muscle. A mutated hGH gene that does not produce active growth hormone was fused to the -3- to +50-kb transferrin sequences to produce the -3-kb mTf-hGX construct. A liver-specific pattern of expression was observed in transgenic mice harboring the -3-kb mTf-hGX construct, and this mutated transgene was shown to be induced four- to sevenfold by either bovine or human growth hormone. These results demonstrate the presence of a growth hormone-responsive element between -3 and +50 kb in the 5'-flanking region of the mTf gene promoter.

Mapping of linear B-cell epitopes of the S2 subunit of pertussis toxin

The linear immunogenic and antigenic structure of the S2 subunit of pertussis toxin was investigated with synthetic peptides corresponding to regions of the protein sequence predicted to contain surface-exposed hydrophilic beta turns. Five peptides as peptide-bovine serum albumin conjugates were recognized by anti-pertussis toxin antiserum and were thus designated "immunogenic epitopes." Two prominent immunogenic epitopes were specified by peptides corresponding to sequences spanning R107-120 and R186-199, whereas peptides corresponding to residues R35-50 and R91-106 were only bound in low titer. Three peptides as thyroglobulin conjugates elicited antisera in rabbits that bound intact pertussis toxin by enzyme-linked immunosorbent assay and immunoblot. These peptides were designated "antigenic epitopes." The most prominent antigenic determinant was localized to the N-terminal end of the S2 sequence encompassing residue R1-7. Peptides R35-50 and R91-106 represented two minor antigenic epitopes. Antisera to two additional peptides corresponding to residues R134-149 and R186-199 recognized the S2 subunit only by Western blotting (immunoblotting). Only antiserum raised against peptide R91-106 also recognized the S3 subunit by Western blotting, indicating a marked antigenic and probably also structural difference between the two highly homologous subunits.

Attempt to visualize an endogenous lectin recognizing α-L-fucose immunohistochemically in the rabbit oviduct

An immunohistochemical study was conducted in order to visualize an endogenous lectin, having specificity for alpha-L-fucose residues, in the ampullary and isthmic tracts of the rabbit oviduct. 2 different sequences (S1 and S2) were tested using antilectin UEA I antibody. Reactive sites towards S2 sequence were localized at ciliated cell level in the ampulla; in the isthmus, instead, the staining was present both in ciliated and secretory cells. A role in the sugar immobilization and gamete and zygote protection was postulated for the endogenous lectin evidenced.