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PubMed1991

Intermolecular hybridization of 5S rRNA with 18S rRNA: identification of a 5'-terminally-located nucleotide sequence in mouse 5S rRNA which base-pairs with two specific complementary sequences in 18S rRNA.

Eukaryotic 5S rRNA hybridizes specifically with 18S rRNA in vitro to form a stable intermolecular RNA:RNA hybrid. We have used 5S rRNA/18S rRNA fragment hybridization studies coupled with ribonuclease digestion and primer extension/chain termination analysis of 5S rRNA:18S rRNA hybrids to more completely map those mouse 5S rRNA and 18S rRNA sequences responsible for duplex formation. Fragment hybridization analysis has defined a 5'-terminal region of 5S rRNA (nucleotides 6-27) which base-pairs with two independent sequences in 18S rRNA designated Regions 1 (nucleotides 1157-1180) and 2 (nucleotides 1324-1339). Ribonuclease digestion of isolated 5S rRNA:18S rRNA hybrids with both single-strand- and double-strand-specific nucleases supports the involvement of this 5'-terminal 5S rRNA sequence in 18S rRNA hybridization. Primer extension/chain termination analysis of isolated 5S rRNA:18S rRNA hybrids confirms the base-pairing of 5S rRNA to the designated Regions 1 and 2 of 18S rRNA. Using these results, 5S rRNA:18S rRNA intermolecular hybrid structures are proposed. Comparative sequence analysis revealed the conservation of these hybrid structures in higher eukaryotes and the same but smaller core hybrid structures in lower eukaryotes and prokaryotes. This suggests that the 5S rRNA:16S/18S rRNA hybrids have been conserved in evolution for ribosome function.

Animals↗
PubMed1992

Identification of a developmentally regulated gene in the mouse central nervous system which encodes a novel proline rich protein.

A full length cDNA whose corresponding mRNA is down-regulated during the mouse embryonic brain development was isolated. The cDNA contains a single long open reading frame which could encode a protein with relative molecular mass of 41 kDa. The predicted gene product contains long stretches of prolines towards the NH2-terminus, followed by a leucine/proline rich region. The cDNA probe detected a number of mRNA species in Northern blot analysis. The reverse transcriptase-polymerase chain reaction analysis of mRNA from adult mouse tissues indicated that heart and testis expressed this gene (named NDPP-1) at relatively high levels, while lower levels of mRNA were detected in a number of other tissues. Expression of NDPP-1 was also detected in embryonic carcinoma and pheochromocytoma cell lines, but not in fibroblasts. The cDNA hybridized to genomic DNA from several vertebrates species in Southern blot analysis indicating interspecies conservation of this gene. The interesting pattern of expression of the NDPP-1 gene during mouse brain development and the structure of its putative protein product indicate that this gene may play an important biological role in the development of mouse central nervous system.

Animals↗
PubMed1992

Marked sequence diversity in the putative envelope proteins of hepatitis C viruses.

The nucleotide sequences of cDNAs (414 base pairs) encoding parts of putative envelope proteins (gp35 and gp70) of 40 isolates of hepatitis C virus (HCV-J) derived from 30 independent plasma or liver specimens from Japanese patients (13 with chronic hepatitis, 14 with hepatocellular carcinoma and 3 hemophiliacs who had received imported clotting factors), were analyzed using the polymerase chain reaction. Approximately 29-38% of the nucleotide sequences of the HCV-J isolates examined differed from those of isolates from the United States (HCV-US). Furthermore, 12-24% and 8-17% sequence diversities were found within the isolates of HCV-J and HCV-US, respectively. The diversities of the amino acid sequences were the same or greater than those of the nucleotide sequences. We confirmed that two hypervariable regions (HVR1 and HVR2) were present in this amplified region, as described in our previous report (Hijikata et al., 1991a) and we found that the HVR1 regions of HCV-J and HCV-US were 27 and 21 amino acids in length, respectively, and began from the N-terminal amino acid of gp70. HVR2 was found in HCV-J, but not in HCV-US isolates, in which the corresponding region of the genome was conserved. During the analysis, plural HCV genomes were found in 6 of 30 specimens. These plural HCV genomes in a single specimen were concluded to be derived from the same HCV ancestor, because of their relative low sequence diversities (about 10% in their nucleotide sequences).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Molecular basis for vaccine development against anaplasmosis and babesiosis.

Immunization of livestock against the erythroparasitic pathogens Anaplasma marginale, Babesia bigemina, and Babesia bovis with safe and effective killed vaccines is not yet feasible on a practical basis. However, the immune protection afforded by recovery from natural infection and premunition indicates that microbial epitopes capable of inducing immunity exist and that the bovine immune system can be primed appropriately. Induction of protection by immunization with killed parasite fractions, enriched for polypeptides with surface exposed epitopes, supports a focus on surface epitopes, including apical complex organellar epitopes in Babesia, for vaccine development. Cloning, sequencing, and expression of genes encoding these key surface polypeptides has allowed examination of polypeptide function and detailed analysis of epitope conservation in light of genetic polymorphism. In this paper, the characterization of these polypeptides at the epitope level and their roles in inducing protective immunity are reviewed. Definition of these epitopes, in combination with improved understanding of immune mechanisms, provides the basis for development of effective recombinant vaccines against anaplasmosis and babesiosis.

Anaplasma↗
PubMed1994

Diversity and relative strength of tandem promoters for the antibiotic-resistance genes of several integrons.

The integron is a new type of mobile element containing one or more antibiotic-resistance-encoding genes site-specifically integrated as cassettes. The integrated genes are expressed from a common promoter region located in an adjacent conserved segment. Sequence analysis has revealed the existence of four versions of the integron promoters. In this study, we have determined the relative strength of the different integron promoters and compared their activity with that of the tac promoter. Each version of the promoter was cloned upstream from a promoter-less chloramphenicol acetyltransferase-encoding gene (cat) in plasmid pKK232-8. CAT activity was used to measure transcriptional expression from the promoters of the antibiotic-resistance operon. The strongest promoter is the version (TTGACAN17TAAACT) found in plasmid R388 and in transposon Tn1696. This promoter is six times more efficient than the derepressed tac promoter.

Acetylation↗
PubMed1990

GAP-43 as a 'calmodulin sponge' and some implications for calcium signalling in axon terminals.

In most neurons, the maturation of axonal growth cones to become stable synaptic terminals is accompanied by a dramatic decline in the abundance of a major growth cone component, GAP-43. Accumulation of GAP-43 persists, however, in a minority of mature synaptic terminals. What properties of axons and their terminals are affected by these changes in GAP-43 expression? Storm and colleagues first noted that the membrane- and calmodulin-binding properties of GAP-43 (a.k.a. P-57 or neuromodulin) could allow it to sequester a large fraction of calmodulin to the submembranous regions, and to release free calmodulin in response to protein kinase C activation. Analysis of evolutionarily conserved sequences in GAP-43 indicates that these properties are central to the biological effects of the protein. If GAP-43 is presumed to inactivate bound calmodulin, the network of GAP-43 in an axon terminal could be considered a regulatable calmodulin buffer, or 'calmodulin sponge', absorbing free calmodulin and releasing it in response to activation of protein kinase C. Such a calmodulin sponge has properties that could be useful in modulating the responses of membrane and cytoskeletal assembly events to calcium signals in growth cones, and in mediating long-term potentiation of neurotransmitter release from some pre-synaptic terminals.

Animals↗
PubMed1995

Comparative analysis of chicken Hoxb-4 regulation in transgenic mice.

We cloned the chicken Hoxb-4 gene and performed in situ analysis to investigate conservation in patterns of expression between the chicken and mouse. The anterior boundaries of expression for both genes in segmented tissues, such as the hindbrain and paraxial mesoderm, map to the same rhombomere (r) (r6/r7) and somite (s) (s6/s7) limits, showing a direct correlation between expression of a specific Hox gene and patterning identical axial structures in both species. Given this similarity in expression we have tested the functional activity of cis-regulatory regions from the chicken Hoxb-4 gene in transgenic mice to identify and map components conserved between the species. We identified enhancers which contain conserved blocks of sequence identity and which are necessary to mediate mesodermal and neural restricted patterns of expression. However, only the neural enhancer directs the proper anterior boundary of expression (r6/r7), indicating that only a subset of the underlying molecular components regulating Hoxb-4 expression are functionally conserved between species.

Animals↗
PubMed2004

Effects of stent length and lesion length on coronary restenosis.

The choice of drug-eluting versus bare metal stents is based on costs and expectations of restenosis and thrombosis risk. Approaches to stent placement vary from covering just the zone of maximal obstruction to stenting well beyond the lesion boundaries (normal-to-normal vessel). The independent effects of stented lesion length, nonstented lesion length, and excess stent length, on coronary restenosis have not been evaluated for bare metal or drug-eluting stents. We analyzed the angiographic follow-up cohort (1,181 patients) from 6 recent bare metal stent trials of de novo lesions in native coronary arteries. Stent length exceeded lesion length in 87% of lesions (mean lesion length 12.4 +/- 6.3 mm, mean stent length 20.0 +/- 7.9 mm, mean difference 7.6 +/- 7.9 mm). At 6- to 9-month follow-up, the mean percent diameter stenosis was 39.1 +/- 20.1%. In an adjusted multivariable model of percent diameter stenosis, each 10 mm of stented lesion length was associated with an absolute increase in percent diameter stenosis of 7.7% (p <0.0001), whereas each 10 mm of excess stent length independently increased percent diameter stenosis by 4.0% (p <0.0001) and increased target lesion revascularization at 9 months (odds ratio 1.12, 95% confidence interval 1.02 to 1.24). Significant nonstented lesion length was uncommon (12.5% of cases). In summary, stent length exceeded lesion length in most stented lesions, and the amount of excess stent length increased the risk of restenosis independent of the stented lesion length. This analysis supports a conservative approach of matching stent length to lesion length to reduce the risk of restenosis with bare metal stents.

Clinical Trials as Topic↗
PubMed2004

Structure of primate and rodent orthologs of the prostate cancer susceptibility gene ELAC2.

The human ELAC2 gene was the first candidate prostate cancer susceptibility gene identified by linkage analysis and positional cloning. DNA sequence indicates a protein of 826 amino acids encoded by 24 exons. In the present study, we characterized the coding sequence of chimpanzee and gorilla ELAC2 orthologs by direct sequencing of genomic fragments, and of cynomolgus monkey and rat orthologs by screening cDNA libraries. The orthologs characterized in the chimpanzee, gorilla and cynomolgus monkey also encode proteins of 826 amino acids, sharing 98.9%, 98.5% and 93.7% sequence identity with the human protein. Our analyses of the mouse ELAC2 gene identified two alternative mRNA transcripts. One is translated into a protein of 824 a.a. (mouse ELAC2), whereas the other one encodes a protein of 831 amino acids (mouse ELAC2A) resulting from an alternatively spliced form of 25 exons. The rat ELAC2 gene ortholog also expressed two similar alternatively spliced transcripts. These two forms are ubiquitously expressed in mouse and rat tissues. The highest levels of expression of the ELAC2 form are observed in the testis while the lowest levels are seen in the prostate and in the muscle. However, it is of interest to note that the relative abundance of the rat and mouse ELAC2 transcripts, measured by real-time quantitative PCR, is higher than the respective ELAC2A forms in all surveyed tissues except for the prostate and the muscle. The ELAC2A transcript levels are 4.1 to 5.0-fold higher than the ELAC2 levels in the prostate of rat and mouse, respectively. A fine analysis of the conserved domains on the primary structure of ELAC2 orthologs revealed the presence of a putative beta-CASP domain shared by the PSO2 (SNM1) DNA interstrand cross-link repair proteins, and the 73-kDa subunit of mRNA 3' end cleavage and polyadenylation specificity factor (CPSF73) as well as Artemis proteins, thus suggesting a potential interaction of ELAC2 gene product with nucleic acids and more specifically with RNA targets. Taken together, these data offer useful tools to further study the regulation and cellular function of ELAC2 gene in experimental models and provide further insight concerning conserved amino acid motifs that could have biological significance.

Alternative Splicing↗
PubMed2005

Evolution and expression of translationally controlled tumour protein (TCTP) of fish.

Translationally Controlled Tumour Protein (TCTP) is one of the abundant and ubiquitously expressed proteins in metazoans. In order to better understand its functions in non-mammalians, cDNA encoding full-length TCTP has been isolated and characterized from a teleost fish, Labeo rohita (rohu). Encoded by a 1043 nucleotide mRNA, rohu TCTP consists of 171 amino acids and is expressed in all organs, except in brain. Secondary structure of fish TCTP mRNAs shows that they could be potential substrates for RNA specific protein kinase PKR. The three-dimensional structure of rohu TCTP has been determined, as the first metazoan model of this protein. The conservational and phylogenic clustering of plant and animal TCTP sequences is consistent with the eukaryotic classification, and is suggestive of early origin for the TCTP ortholog in eukaryote evolution, as early as 1.0 x 10(9) years ago. Despite significant conservation, meticulous sequence analysis reveals striking differences that suggest possible new functions for TCTP.

Amino Acid Sequence↗
PubMed2005

Cloning and characterization of Xenopus beta2-microglobulin.

cDNAs for Xenopus beta2-microglobulin (beta2m), the obligatory light chain of most vertebrate Major Histocompatibility Complex (MHC) class I molecules, were isolated and ESTs were identified. Alignment of the deduced amino acid sequence to other species' beta2m showed that the overall structure is evolutionarily conserved, and phylogenetic analysis showed that the Xenopus beta2m sequence is intermediate between fish and bird/mammal beta2m. The Xenopus beta2m mRNA is expressed ubiquitously with highest expression in intestine, spleen, and thymus, correlating well with classical class Ia expression. beta2m mRNA and protein were also detected in Xenopus thymic tumor and kidney cell lines. Segregation analysis on a tetraploid Xenopus laevis family revealed two independently segregating, non-MHC-linked loci. As expected, only one locus was found in the diploid Xenopus tropicalis, strongly suggesting that the two beta2m loci in the tetraploid species were generated by genome-wide duplication, and did not undergo diploidization unlike many other MHC genes.

Amino Acid Sequence↗
PubMed2006

Identification and functional analysis of bifunctional ent-kaurene synthase from the moss Physcomitrella patens.

ent-Kaurene is the key intermediate in biosynthesis of gibberellins (GAs), plant hormones. In higher plants, ent-kaurene is synthesized successively by copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) from geranylgeranyl diphosphate (GGDP). On the other hand, fungal ent-kaurene synthases are bifunctional cyclases with both CPS and KS activity in a single polypeptide. The moss Physcomitrella patens is a model organism for the study of genetics and development in an early land plant. We identified ent-kaurene synthase (PpCPS/KS) from P. patens and analyzed its function. PpCPS/KS cDNA encodes a 101-kDa polypeptide, and shows high similarity with CPSs and abietadiene synthase from higher plants. PpCPS/KS is a bifunctional cyclase and, like fungal CPS/KS, directly synthesizes the ent-kaurene skeleton from GGDP. PpCPS/KS has two aspartate-rich DVDD and DDYFD motifs observed in CPS and KS, respectively. The mutational analysis of two conserved motifs in PpCPS/KS indicated that the DVDD motif is responsible for CPS activity (GGDP to CDP) and the DDYFD motif for KS activity (CDP to ent-kaurene and ent-16alpha-hydroxykaurene).

Alkyl and Aryl Transferases↗
PubMed2004

Expression of proprotein convertase 2 mRNA in the ovarian follicles of the medaka, Oryzias latipes.

Proprotein convertases (PCs) are enzymes responsible for processing the precursors of many bioactive peptides in vertebrates and invertebrates. In the present study, a cDNA for proprotein convertase 2 (PC2) was cloned for the first time from a fish. The clone, which was isolated from the ovary of the medaka, Oryzias latipes, by a combination of RT-PCR cloning and 5'- and 3'-rapid amplification of cDNA ends, codes for a protein of 641 amino acid residues highly homologous to other vertebrate PC2. The medaka preproPC2 consists of a signal sequence, a propeptide with sites for autocatalytic activation, a Kex2-like catalytic domain, and a P-domain. The catalytic triad residues (Asp-169, His-210, and Ser-386) were all conserved. Northern blot analysis revealed that PC2 was expressed in the brain, ovary, and kidney of the fish. The size of PC2 mRNA expressed in the ovary was 2.3 kb, whereas those of the brain and kidney were 2.8 kb. This size difference was attributed to the lack of an approximately 300-bp nucleotide sequence just before the poly(A)+ tail of the ovarian PC2 mRNA. Ovarian expression of the PC2 gene was found in the medaka but not in the mouse, and therefore further analysis was conducted for the fish ovary. The greatest expression of PC2 mRNA in the oocytes of small growing follicles in the mature medaka was demonstrated by Northern blotting, RT-PCR and in situ hybridization analysis. These results suggest that PC2 may play a role in the processing of proproteins and/or pro-hormones expressed in the growing oocytes.

Amino Acid Sequence↗
PubMed2006

Structural and biochemical study of effector molecule recognition by the E.coli glyoxylate and allantoin utilization regulatory protein AllR.

The interaction of Escherichia coli AllR regulator with operator DNA is disrupted by the effector molecule glyoxylate. This is a general, yet uncharacterized regulatory mechanism for the large IclR family of transcriptional regulators to which AllR belongs. The crystal structures of the C-terminal effector-binding domain of AllR regulator and its complex with glyoxylate were determined at 1.7 and 1.8 A, respectively. Residues involved in glyoxylate binding were explored in vitro and in vivo. Altering the residues Cys217, Ser234 and Ser236 resulted in glyoxylate-independent repression by AllR. Sequence analysis revealed low conservation of amino acid residues participating in effector binding among IclR regulators, which reflects potential chemical diversity of effector molecules, recognized by members of this family. Comparing the AllR structure to that of Thermotoga maritima TM0065, the other representative of the IclR family that has been structurally characterized, indicates that both proteins assume similar quaternary structures as a dimer of dimers. Mutations in the tetramerization region, which in AllR involve the Cys135-Cys142 region, resulted in dissociation of AllR tetramer to dimers in vitro and were functionally inactive in vivo. Glyoxylate does not appear to function through the inhibition of tetramerization. Using sedimentation velocity, glyoxylate was shown to conformationally change the AllR tetramer as well as monomer and dimer resulting in altered outline of AllR molecules.

Allantoin↗
PubMed2006

Desulfovibrio desulfuricans G20 tetraheme cytochrome structure at 1.5 Angstrom and cytochrome interaction with metal complexes.

The structure of the type I tetraheme cytochrome c(3) from Desulfovibrio desulfuricans G20 was determined to 1.5 Angstrom by X-ray crystallography. In addition to the oxidized form, the structure of the molybdate-bound form of the protein was determined from oxidized crystals soaked in sodium molybdate. Only small structural shifts were obtained with metal binding, consistent with the remarkable structural stability of this protein. In vitro experiments with pure cytochrome showed that molybdate could oxidize the reduced cytochrome, although not as rapidly as U(VI) present as uranyl acetate. Alterations in the overall conformation and thermostability of the metal-oxidized protein were investigated by circular dichroism studies. Again, only small changes in protein structure were documented. The location of the molybdate ion near heme IV in the crystal structure suggested heme IV as the site of electron exit from the reduced cytochrome and implicated Lys14 and Lys56 in binding. Analysis of structurally conserved water molecules in type I cytochrome c(3) crystal structures identified interactions predicted to be important for protein stability and possibly for intramolecular electron transfer among heme molecules.

Binding Sites↗
PubMed2006

Engineering, biophysical characterisation and binding properties of a soluble mutant form of annexin A2 domain IV that adopts a partially folded conformation.

The four approximately 75-residue domains (repeats) that constitute the annexin core structure all possess an identical five-alpha-helix bundle topology, but the physico-chemical properties of the isolated domains are different. Domain IV of the annexins has previously been expressed only as inclusion bodies, resistant to solubilisation. Analysis of the conserved, exposed hydrophobic residues of the four annexin domains reveals that domain IV contains the largest number of hydrophobic residues involved in interfacial contacts with the other domains. We designed five constructs of domain IV of annexin A2 in which several interfacial hydrophobic residues were substituted by hydrophilic residues. The mutant domain, in which all fully exposed hydrophobic interfacial residues were substituted, was isolated as a soluble protein. Circular dichroism measurements indicate that it harbours a high content of alpha-helical secondary structure and some tertiary structure. The CD-monitored (lambda=222 nm) thermal melting profile suggests a weak cooperative transition. Nuclear magnetic resonance (1H-15N) correlation spectroscopy reveals heterogeneous line broadening and an intermediate spectral dispersion. These properties are indicative of a partially folded protein in which some residues are in a fairly structured conformation, whereas others are in an unfolded state. This conclusion is corroborated by 1-anilinonaphthalene-8-sulfonate fluorescence (ANS) analyses. Surface plasmon resonance measurements also indicate that this domain binds heparin, a known ligand of domain IV in the full-length annexin A2, although with lower affinity.

Amino Acid Sequence↗
PubMed2006

Structural analysis of the plakin domain of bullous pemphigoid antigen1 (BPAG1) suggests that plakins are members of the spectrin superfamily.

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins. The plakins are multi-domain proteins that have been shown to interact with microtubules, actin filaments and intermediate filaments, as well as proteins found in cellular junctions. These interactions are mediated through different domains on the plakins. The interactions between plakins and components of specialized cell junctions such as desmosomes and hemidesmosomes are mediated through the so-called plakin domain, which is a common feature of the plakins. We report the crystal structure of a stable fragment from BPAG1, residues 226-448, defined by limited proteolysis of the whole plakin domain. The structure, determined by single-wavelength anomalous diffraction phasing from a selenomethionine-substituted crystal at 3.0 A resolution, reveals a tandem pair of triple helical bundles closely related to spectrin repeats. Based on this structure and analysis of sequence conservation, we propose that the architecture of plakin domains is defined by two pairs of spectrin repeats interrupted by a putative Src-Homology 3 (SH3) domain.

Amino Acid Sequence↗
PubMed2005

Rapid subgroup identification of the flaviviruses using degenerate primer E-gene RT-PCR and site specific restriction enzyme analysis.

A simplified and rapid method for the diagnosis of all flaviviruses could provide an important tool for understanding their epidemiology. A protocol based on the use of degenerate nested oligonucleotide primers and RT-PCR was developed for the identification of flaviviruses. The primers were selected to flank the three E-gene markers that identify the viruses, giving DNA products of 971-986 (outer primers) and 859-884 bp (inner primers). Eighty five percent of E genes from flaviviruses representing most of the genus were specifically amplified, representing viruses from each of the 14 virus groups defined by the seventh International Committee for the Taxonomy of Viruses. Categorisation of the flavivirus cDNA products into the corresponding virus groups was undertaken through restriction enzyme analysis by defining conserved restriction sites common to related viruses in appropriate virus groups. Ninety percent of the known vector-borne flaviviruses with published full length E-gene sequences could be identified within 10 h.

Animals↗