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PubMed2002

Sequence conservation and distribution of the fusobacterial immunosuppressive protein gene, fipA.

Fusobacterium nucleatum is a gram-negative anaerobe involved in various diseases, including periodontitis. Recently, other investigators isolated the F. nucleatum FDC 364 fusobacterial immunosuppressive protein (FIP). One subunit, FipA, impairs T-cell activation in vitro and shows homology with beta-ketothiolases. However, its distribution and variability among fusobacteria was not reported. Cloned fipA gene sequences from F. nucleatum ssp. polymorphum (ATCC 10953) and F. nucleatum ssp. nucleatum (ATCC 23726) shared 89 and 92% identity, respectively, with FDC 364 fipA, and 90 and 94% identity, respectively, with the FDC 364 FipA predicted amino acid sequence. Southern blot analyses of chromosomal DNA from fusobacterial strains, including F. nucleatum and other Fusobacterium species, were performed using partial fipA sequences as probes. The results indicate that fipA is highly conserved among the F. nucleatum strains examined and that fipA homologues are widely distributed among fusobacteria. A clear relationship between immune suppression, metabolism and the FipA protein remains to be determined.

Acetyl-CoA C-Acetyltransferase↗

PubMed2002

Peptomics, identification of novel cationic Arabidopsis peptides with conserved sequence motifs.

Few plant peptides involved in intercellular communication have been experimentally isolated. Sequence analysis of the Arabidopsis thaliana genome has revealed numerous transmembrane receptors predicted to bind proteinacious ligands, emphasizing the importance of identifying peptides with signaling function. Annotation of the Arabidopsis genome sequence has made it possible to identify peptide-encoding genes. However, such annotational identification is impeded because small genes are poorly predicted by gene-prediction algorithms, thus prompting the alternative approaches described here. We initially performed a systematic analysis of short polypeptides encoded by annotated genes on two Arabidopsis chromosomes using SignalP to identify potentially secreted peptides. Subsequent homology searches with selected, putatively secreted peptides, led to the identification of a potential, large Arabidopsis family of 34 genes. The predicted peptides are characterized by a conserved C-terminal sequence motif and additional primary structure conservation in a core region. The majority of these genes had not previously been annotated. A subset of the predicted peptides show high overall sequence similarity to Rapid Alkalinization Factor (RALF), a peptide isolated from tobacco. We therefore refer to this peptide family as RALFL for RALF-Like. RT-PCR analysis confirmed that several of the Arabidopsis genes are expressed and that their expression patterns vary. The identification of a large gene family in the genome of the model organism Arabidopsis thaliana demonstrates that a combination of systematic analysis and homology searching can contribute to peptide discovery.

Algorithms↗

PubMed1995

Amplification of bacterial DNA using highly conserved sequences: automated analysis and potential for molecular triage of sepsis.

OBJECTIVE: The clinical diagnosis of sepsis remains difficult, particularly in the young child, and would be improved by a rapid and reliable method for identification of bacteria in blood and other body fluids. Polymerase chain reaction (PCR) amplification of highly conserved DNA sequences found in all bacteria would permit fast and sensitive determination of the presence of bacteria in clinical specimens. METHODOLOGY: A primer pair for highly conserved regions of bacterial DNA encoding 16S ribosomal RNA (rDNA) was utilized for PCR amplification. PCR products were analyzed by gel electrophoresis, and, after modification of the primers, by an automated 96-well plate reader. RESULTS: rDNA was amplified from 12 different species of bacteria, including Gram-negative and -positive organisms. No signal was observed when total human DNA was used as template. Colorimetric analysis of amplified sequences using a 96-well format was also successful. CONCLUSIONS: We conclude that a single primer pair designed to anneal to a highly conserved region of bacterial DNA can amplify DNA specimens from a variety of different bacteria, while not amplifying human DNA. Such a molecular genetics approach can be fully automated with existing robotic technology. Because of speed, sensitivity, and cost, molecular triage of patients with signs and symptoms of possible bacterial infection will decrease morbidity and mortality among those with unrecognized bacteremia who are managed as outpatients, and will dramatically reduce hospital expenses from individuals who are admitted and are not bacteremic.

Autoanalysis↗

PubMed1996

RER, an evolutionarily conserved sequence upstream of the rhodopsin gene, has enhancer activity.

Previous transgenic mouse experiments localized the mammalian rhodopsin gene promoter to a region just upstream of the mRNA start site, and also suggested the existence of a second more distal regulatory region. A highly conserved 100-base pair (bp) sequence which is homologous to the red and green opsin locus control region is located 1.5-2 kilobases upstream of the rhodopsin gene (depending on the species). In order to test the activity of this 100-bp region, transgenic mice were generated with bovine rhodopsin promoter/lacZ constructs which differed only by the presence or absence of the sequence. Of 11 lines generated, all demonstrated photoreceptor-specific expression of the transgene, but the lines with the putative regulatory region showed significantly higher expression. Additional transgenic lines in which the region was fused to a minimal heterologous promoter did not show transgene expression in the retina. Gel mobility shift and DNase I footprint assays demonstrated that bovine retinal nuclear extracts contain retina-specific as well as ubiquitously expressed factors that interact with the putative regulatory region in a sequence-specific manner. These results indicate that the 100-bp sequence can indeed function in vivo as a rhodopsin enhancer region.

Animals↗

PubMed1988

The complete nucleotide sequence of the outer coat protein, VP5, of the Australian bluetongue virus (BTV) serotype 1 reveals conserved and non-conserved sequences.

The complete sequence of the outer coat protein, VP5, of the Australian BTV serotype 1 was determined and found to be 1634 nucleotides in length. One single open reading frame of 526 amino acids was observed defining a protein of Mr 59,252 and having a charge of +0.5 at neutral pH. When compared to VP5 of BTV serotype 10 from the United States of America (US) (Purdy et al., 1986, J. Gen. Virol. 67, 957) a homology of 68% at the nucleotide level and 76% at the amino acid level, was observed. However, this conservation at the protein level was more apparent in certain regions of the gene. In four main regions the conservation varied from 83-91% while in the remaining regions the homology dropped to between 56-62%. Many of the amino acid substitutions were conservative in nature, raising the apparent overall homology to 87%. Comparisons of the hydropathy profiles of the two proteins again revealed a remarkable degree of conservation. The importance of these observations is discussed.

Amino Acid Sequence↗

PubMed2000

Opsonic potential, protective capacity, and sequence conservation of the Treponema pallidum subspecies pallidum Tp92.

By means of a differential screening technique, a 92-kDa antigen, designated Tp92, was identified from Treponema pallidum subspecies pallidum. This protein is similar in sequence to the protective surface antigens D15 from Haemophilus influenzae and Oma87 from Pasteurella multocida. Amino acid sequence analyses revealed a cleavable N-terminal signal sequence and predicted the outer membrane location for Tp92. In support of this, antiserum raised against recombinant Tp92 promotes opsonization and phagocytosis of T. pallidum by rabbit macrophages, and anti-Tp92 reactivity is absent from washed treponemal preparations presumed to be lacking outer membranes. The Tp92 amino acid sequence is 95.5%-100% conserved among 11 strains representing 4 pathogenic treponemes, and immunization with recombinant Tp92 partially protected rabbits from subsequent T. pallidum challenge. These results demonstrate that Tp92 is an invariant, immunoprotective antigen that may be present on the surface of T. pallidum and may represent a potential vaccine candidate for syphilis.

Amino Acid Sequence↗

PubMed2004

Evolutionarily conserved sequence elements that positively regulate IFN-gamma expression in T cells.

Our understanding of mechanisms by which the expression of IFN-gamma is regulated is limited. Herein, we identify two evolutionarily conserved noncoding sequence elements (IFNgCNS1 and IFNg CNS2) located approximately 5 kb upstream and approximately 18 kb downstream of the initiation codon of the murine Ifng gene. When linked to the murine Ifng gene (-3.4 to +5.6 kb) and transiently transfected into EL-4 cells, these elements clearly enhanced IFN-gamma expression in response to ionomycin and phorbol 12-myristate 13-acetate and weakly enhanced expression in response to T-bet. A DNase I hypersensitive site and extragenic transcripts at IFNgCNS2 correlated positively with the capacity of primary T cell subsets to produce IFN-gamma. Transcriptionally favorable histone modifications in the Ifng promoter, intronic regions, IFNgCNS2, and, although less pronounced, IFNgCNS1 increased as naïve T cells differentiated into IFN-gamma-producing effector CD8+ and T helper (TH) 1 T cells, but not into TH2 T cells. Like IFN-gamma expression, these histone modifications were T-bet-dependent in CD4+ cells, but not CD8+ T cells. These findings define two distal regulatory elements associated with T cell subset-specific IFN-gamma expression.

Animals↗

PubMed2004

Sequence, conservation, and quantitative expression of rainbow trout Myf5.

The success of rainbow trout as an aquaculture species is dependent on the ability to produce fish with large amounts of high-quality lean muscle. It is therefore important to understand not only the best conditions under which to raise the fish but also the molecular control of muscle growth. Vertebrate muscle growth is initiated by the specification of myogenic precursor cells into myoblasts. The myoblasts proliferate and fuse to form multinucleated myotubes, which mature into myofibers. A family of basic helix-loop-helix (bHLH) transcription factors, the Myogenic Regulatory Factors (MRFs), controls these events. In trout, two MRF-encoding genes, TMyoD (of which there are two) and Tmyogenin, have been identified. However, the primary MRF-encoding Myf5 is not yet sequenced. Here, using degenerate PCR and 5' and 3' RACE, the cDNA sequence of trout Myf5 (TMyf5) is identified. Translation of the cDNA reveals that TMyf5 is a bHLH protein with homology to Myf5 and MRFs in other organisms. It is expressed mainly in red and white muscle, suggesting that it shares functional homology to Myf5 in other species. The molecular control of muscle growth has been well-characterized in mammals, but there are differences in the growth of fish muscle, highlighting the need for characterization of MRFs in fish species, particularly those in which understanding muscle growth will have a positive impact on the economic potential of the species.

Amino Acid Sequence↗

PubMed1992

A micronucleus-limited sequence family in Tetrahymena thermophila: organization and sequence conservation.

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, sequence reorganization including sequence loss occurs. Addressing questions about the organization and nucleotide sequence of micronucleus limited regions can lead to insights about mechanisms of DNA rearrangements during macronuclear development as well as mechanisms for the maintenance of the stability of micronucleus-limited sequence families. We have previously identified a moderately repetitive micronucleus-limited sequence family called X-H (family members hybridize to an approximately 450 bp Xbal-HindIII restriction fragment), completely absent from macronuclear DNA. The first member of this family which we isolated is associated with terminal sequences characteristic of a Tel-1 element, a putative micronuclear transposable element. Two additional family members have been isolated which are not closely associated with Tel-1 terminal sequences. We have nucleotide sequence data for three cloned members of the X-H family. This analysis has demonstrated that the longest cloned members of the X-H family share a region of homology of approximately 2,400 bp and are highly conserved, differing only by small insertions or deletions of 100 bp or less. The sequences from one of the sequenced family members flanking the region of homology are themselves mostly micronucleus-limited.

Animals↗

PubMed1991

Complete genomic sequence of the murine low affinity Fc receptor for IgE. Demonstration of alternative transcripts and conserved sequence elements.

The complete sequence of the murine low affinity Fc receptor for IgE (Fc epsilon RII), including the 5' and 3' flanking sequences, is reported. The murine Fc epsilon RII gene spans 12.9 kb and includes 12 exons surrounding 11 introns. The composite exon sequence is virtually identical to previously reported murine Fc epsilon RII cDNA sequences. Much of the proximal promoter regions of the mouse and human homologues of Fc epsilon RII show remarkable homology to each other, including three promoter elements previously identified for MHC class II genes. The reported exon/intron structure of the human FC epsilon RII is similar to the murine homologue, except that the latter has an additional exon coding for a fourth amino acid repetitive sequence (vs three in the human gene). RNase protection studies have identified an additional transcript within intron 2 of murine Fc epsilon RIIa, similar to the human Fc epsilon RIIb form but with a different predicted sequence of the first six amino acids. This transcript is present in the mRNA of purified splenic B cells, but not in the mRNA of the Fc epsilon RII+ B lymphoma cell line M12.4.5. The murine Fc epsilon RII gene contains a large intron (4.2 kb) separating the lectin and nonlectin coding regions, and several repetitive sequences are found clustered within this intron. These results emphasize the importance of the demarcation between these domains and allude to their evolutionary and functional significance.

Amino Acid Sequence↗

PubMed1998

Chemotactic adaptation is altered by changes in the carboxy-terminal sequence conserved among the major methyl-accepting chemoreceptors.

In Escherichia coli and Salmonella typhimurium, methylation and demethylation of receptors are responsible for chemotactic adaptation and are catalyzed by the methyltransferase CheR and the methylesterase CheB, respectively. Among the chemoreceptors of these species, Tsr, Tar, and Tcp have a well-conserved carboxy-terminal motif (NWET/SF) that is absent in Trg and Tap. When they are expressed as sole chemoreceptors, Tsr, Tar, and Tcp support good adaptation, but Trg and Tap are poorly methylated and supported only weak adaptation. It was recently discovered that CheR binds to the NWETF sequence of Tsr in vitro. To examine the physiological significance of this binding, we characterized mutant receptors in which this pentapeptide sequence was altered. C-terminally-mutated Tar and Tcp expressed in a receptorless E. coli strain mediated responses to aspartate and citrate, respectively, but their adaptation abilities were severely impaired. Their expression levels and attractant-sensing abilities were similar to those of the wild-type receptors, but the methylation levels of the mutant receptors increased only slightly upon addition of attractants. When CheR was overproduced, both the adaptation and methylation profiles of the mutant Tar receptor became comparable to those of wild-type Tar. Furthermore, overproduction of CheR also enhanced adaptive methylation of wild-type Trg, which lacks the NWETF sequence, in the absence of any other chemoreceptor. These results suggest that the pentapeptide sequence facilitates effective adaptation and methylation by recruiting CheR.

Adaptation, Physiological↗

PubMed1994

A conserved sequence motif within the exceptionally diverse telomeric sequences of budding yeasts.

Telomeric DNA sequences have generally been found to be remarkably conserved in evolution, typically consisting of repeated, very short sequence units containing clusters of G residues. Recently however the telomeric DNA of the asexual yeast Candida albicans was shown to consist of much longer repeat units. Here we report the identification of seven additional telomeric sequences from sexual and asexual budding yeast species. The telomeric repeat units from this group of relatively closely related species show more phylogenetic diversity in length (8-25 bp), sequence, and composition than has been seen previously throughout a wide phylogenetic range of other eukaryotes. We also show that certain strains of the asexual diploid species Candida tropicalis have two forms of telomeric repeats, which appear to differ by a single base pair. Despite their great diversity, the telomeric repeat units of C. albicans, Saccharomyces cerevisiae, and all of the species we have examined in this report share a conserved approximately 6-bp motif of T and G residues resembling more typical telomeric sequences.

Base Sequence↗

PubMed1997

Subunit interactions in ABC transporters: a conserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits.

The cytoplasmic membrane proteins of bacterial binding protein-dependent transporters belong to the superfamily of ABC transporters. The hydrophobic proteins display a conserved, at least 20 amino acid EAA---G---------I-LP region exposed in the cytosol, the EAA region. We mutagenized the EAA regions of MalF and MalG proteins of the Escherichia coli maltose transport system. Substitutions at the same positions in MalF and MalG have different phenotypes, indicating that EAA regions do not act symmetrically. Mutations in malG or malF that slightly affect or do not affect transport, determine a completely defective phenotype when present together. This suggests that EAA regions of MalF and MalG may interact during transport. Maltose-negative mutants fall into two categories with respect to the cellular localization of the MalK ATPase: in the first, MalK is membrane-bound, as in wild-type strains, while in the second, it is cytosolic, as in strains deleted in the malF and malG genes. From maltose-negative mutants of the two categories, we isolated suppressor mutations within malK that restore transport. They map mainly in the putative helical domain of MalK, suggesting that EAA regions may constitute a recognition site for the ABC ATPase helical domain.

ATP-Binding Cassette Transporters↗

PubMed1987

A highly conserved sequence in H1 histone genes as an oligonucleotide hybridization probe: isolation and sequence of a duck H1 gene.

A 3.5-kb HindIII fragment of a histone gene cluster was isolated from a recombinant phage out of a duck genomic library. This DNA contains a duck H1 gene and its flanking sequences. The hybridization probe, which was used to screen for the H1 gene, had been designed on the basis of a comparative analysis of available H1 gene and protein data. Most H1 histones contain repeated motifs in their C-terminal domain, and these form part of an octapeptide (ser pro lys lys ala lys lys pro) that is highly conserved in many H1 histone proteins. A comparison of the duck H1 described here with two different published chicken H1 histone sequences reveals conservative amino acid exchanges at 22 (of 217 and 218, respectively) positions. The homology is maintained at the flanking sequences, and includes the putative H1 histone gene-specific signal structures and the established 3' stem and loop structures and the CAAGA box. The duck H1 gene and its flanking sequence have been found in identical arrangements in two recombinant bacteriophages, but minor sequence variations and genomic Southern blotting after HindIII digestion suggest that we have either isolated alleles of this genome segment or that the gene described may occur twice per haploid duck genome.

Amino Acid Sequence↗

PubMed1999

Evolution of ITS1 rDNA in the Digenea (Platyhelminthes: trematoda): 3' end sequence conservation and its phylogenetic utility.

A comparison of ribosomal internal transcribed spacer 1 (ITS1) elements of digenetic trematodes (Platyhelminthes) including unidentified digeneans isolated from Cyathura carinata (Crustacea: Isopoda) revealed DNA sequence similarities at more than half of the spacer at its 3' end. Primary sequence similarity was shown to be associated with secondary structure conservation, which suggested that similarity is due to identity by descent and not chance. Using an analysis of apomorphies, the sequence data were shown to produce a distinct phylogenetic signal. This was confirmed by the consistency of results of different tree reconstruction methods such as distance approaches, maximum parsimony, and maximum likelihood. Morphological evidence additionally supported the phylogenetic tree based on ITS1 data and the inferred phylogenetic position of the unidentified digeneans of C. carinata met the expectations from known trematode life-cycle patterns. Although ribosomal ITS1 elements are generally believed to be too variable for phylogenetic analysis above the species or genus level, the overall consistency of the results of this study strongly suggests that this is not the case in digenetic trematodes. Here, 3' end ITS1 sequence data seem to provide a valuable tool for elucidating phylogenetic relationships of a broad range of phylogenetically distinct taxa.

Animals↗

PubMed1986

Conserved sequences and transcription of the hsp70 gene family in Trypanosoma brucei.

Five Trypanosoma brucei 70-kilodalton heat shock protein-encoding genes (hsp70 genes) were found to be arranged in a tandem array. These hsp70 genes are separated by highly conserved intergenic region sequences of 200 base pairs for one gene and 234 base pairs for the other four genes. This intergenic region sequence is also present in front of the first gene of the tandem array, though at a further distance. All five conserved intergenic regions have sequences that are homologous to the eucaryotic control elements, essential for temperature-induced initiation of transcription by polymerase II. In addition, there is a T-rich region at the 3' end of the hsp70 genes which is homologous to the site of transcription termination of mini-exon genes. Immediately 3' of a putative TATA box, a branch point consensus sequence and six sequences homologous to known trypanosome 3' splice sites were found. It is therefore possible that a PolII promoter is present in the intergenic region sequence. Addition of the 35-nucleotide mini-exon to the hsp70 transcript could thus be mediated by bimolecular splicing. The importance of temperature control for development was illustrated by the response of variant surface glycoprotein-encoding genes to heat shock.

Amino Acid Sequence↗

PubMed1996

Testosterone regulates tissue-specific changes in the binding of a 47-kilodalton protein to a highly conserved sequence in the 3' untranslated region of epidermal growth factor messenger ribonucleic acid.

Epidermal growth factor (EGF) transcripts that use the terminal polyadenylation signal display a dramatic sex difference in the pattern of polyadenylation in the murine submaxillary gland (SMG), whereas those in the kidney do not. It takes 3 days before testosterone treatment begins to change the polyadenylation pattern in female SMG to resemble the male pattern, a finding that supports previous suggestions that posttranscriptional mechanisms are involved in regulating EGF expression. The conservation of a unique 23-b sequence centered on the terminal polyadenylation signal in all published mammalian EGF sequences suggested that trans-acting factors involved in EGF messenger RNA (mRNA) metabolism might bind to this sequence. To investigate this, we prepared 32P-RNA containing the 3' terminal EGF 23-b sequence plus a short poly-A tail, and incubated it with SMG cytosol. Cytosol retarded the electrophoretic mobility of this RNA as a single prominent band on 8% PAGE, and by UV-cross-linking, a single prominent 47-kDa protein was detected on 10% SDS-PAGE. Trypsin abolished both the gel-retarding and cross-linking activities. Cytosol from female SMGs contained approximately 8 times more of both the RNA binding activities than male cytosol. Injecting testosterone (200 microg QOD) into female mice altered both the RNA binding activities in a biphasic fashion, initially increasing them by about 40% at 2 days, then decreasing them by about 65% > or = 5 days, reaching male levels. Kidney cytosol contained both RNA binding activities but displayed neither sexual dimorphism nor testosterone-responsiveness. The tissue-specific testosterone-dependent changes observed in the 47-kDa protein occur before the increase in EGF mRNA levels and before the change in EGF mRNA polyad-enylation, so this cytosolic protein could be a trans-acting factor involved in EGF polyadenylation.

Alkaline Phosphatase↗

PubMed1990

Molecular evolution of class A beta-lactamases: phylogeny and patterns of sequence conservation.

We present a multiple alignment of the amino acid sequences of eight class A beta-lactamases and utilized it to propose a phylogeny, based on the nucleotide sequences of their corresponding genes. We have also used the alignment, together with the alpha-carbon co-ordinates of the Staphylococcus aureus protein, to search systematically for neighbouring residues that share the same pattern of conservation among the different members of the protein family. The distribution of invariant residues and of groups of residues with co-ordinate changes map, predominantly, at the region of the active site and at interfaces between structural elements, respectively. We have also contrasted the distribution of conserved residues with the positions which are known to differ in mutants and variants of class A beta-lactamases.

Amino Acid Sequence↗