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PubMed1992

TATA box-independent transcription of the human tissue plasminogen activator gene initiates within a sequence conserved in related genes.

Transcription of the human tissue-type plasminogen activator (tPA) gene has been reported to initiate from a single site proximal to a TATA box motif [1985, J. Biol. Chem. 260, 11223-11230]. In this study, we utilized primer extension analysis to evaluate the tPA mRNA start site in phorbol-12-myristate 13-acetate (PMA) induced WI-38 human lung fibroblast cells. Whilst some tPA mRNA initiated from the predicted TATA-proximal location (+1), a 10-fold greater proportion of tPA mRNA transcripts initiated 110 bases downstream from a sequence conserved and utilized as the TATA-independent transcription start site in the rodent tPA genes. Moreover, the transfection and expression in different cell types of a cosmid containing the entire human tPA gene resulted in utilization of the same downstream (+110) start site. We propose that this, rather than the previously published position, is the major transcriptional initiation point for the human tPA gene. A core sequence (5'-CAGAGCTG-3') was identified which is common to the TATA-independent mRNA start sites of the human, mouse and rat tPA genes, and which demonstrates only partial similarity to sequences found at the initiation point of other TATA-independent genes.

Amino Acid Sequence↗

PubMed1990

Subrepeats result from regional DNA sequence conservation in tandem repeats in Chironomus telomeres.

Repeat units, widespread in eukaryotic genomes, are often partially or entirely built up of subrepeats. Homogenization between whole repeat units arranged in tandem usually can best be understood as a result of unequal crossing over. Such a mechanism is less plausible for maintaining similarities between subrepeats within a repeat unit when present in a regular array. In Chironomus telomeres, large blocks of tandemly repeated approximately 350 base-pair units contain two or three pairs of subrepeats with high mutual identities, embedded in linker DNA, non-repetitive within the repeat unit. Measurements of evolutionary base changes in two closely related species, Chironomus tentans and Chironomus pallidivittatus, permit us to conclude that the subrepeat arrangement is best explained as a consequence of regional sequence conservation after an earlier duplication of an ancestral half-unit.

Animals↗

PubMed1985

Antibodies to peptides corresponding to a conserved sequence of gonococcal pilins block bacterial adhesion.

Antisera generated against each of seven synthetic peptides corresponding to constant and variable sequences of the pilin from gonococcal strain MS11 were assayed for their ability to crossreact with intact pili from both homologous and heterologous strains. The peptides elicited roughly equal antipeptide responses but varied substantially in their ability to elicit antisera that crossreacted with intact pili. Of the antisera to peptides corresponding to regions of conserved sequence, antisera directed against residues 69-84 were the most efficient in binding pili from all strains tested in both solid-phase assays and immunoblots. Anti-69-84 also efficiently precipitated a tryptic fragment of pilin known to bind human endocervical cells. Sera against the two peptides (121-134 and 135-151) previously shown to contain strain-specific epitopes crossreacted with MS11 pili equally well, but differed in their ability to bind pili from heterologous strains. Anti-121-134 was strain-specific whereas anti-135-151 bound all pilin tested. Each of the sera was examined for its ability to inhibit bacterial adhesion to a human endometrial carcinoma cell line. Sera generated against residues 41-50 and 69-84 successfully inhibited a heterologous gonococcal strain from binding. These peptides could be important components of an effective vaccine for the prevention of gonorrhea.

Adhesiveness↗

PubMed2003

The fully conserved Asp residue in conserved sequence region I of the alpha-amylase family is crucial for the catalytic site architecture and activity.

The alpha-amylase family is a large group of starch processing enzymes [Svensson, B. (1994) Plant Mol. Biol. 25, 141-157]. It is characterized by four short sequence motifs that contain the seven fully conserved amino acid residues in this family: two catalytic carboxylic acid residues and four substrate binding residues. The seventh conserved residue (Asp135) has no direct interactions with either substrates or products, but it is hydrogen-bonded to Arg227, which does bind the substrate in the catalytic site. Using cyclodextrin glycosyltransferase as an example, this paper provides for the first time definite biochemical and structural evidence that Asp135 is required for the proper conformation of several catalytic site residues and therefore for activity.

Aspartic Acid↗

PubMed1999

Seminal-type ribonuclease genes in ruminants, sequence conservation without protein expression?

Bovine seminal ribonuclease (BS-RNase) is an interesting enzyme both for functional and structural reasons. The enzyme is the product of a gene duplication that occurred in an ancestral ruminant. It is possible to demonstrate the presence of seminal-type genes in all other investigated ruminant species, but they are not expressed and show features of pseudogenes. In this paper we report the determination of two pancreatic and one seminal-type ribonuclease gene sequences of swamp-type water buffalo (Bubalus bubalis). The two pancreatic sequences encode proteins with identical amino acid sequences as previously determined for the enzymes isolated from swamp-type and river-type water buffalo, respectively. The seminal-type sequence has no pseudogene features and codes for an enzyme with no unusual features compared with the active bovine enzyme, except for the replacement of one of the cysteines which takes part in the two intersubunit disulfide bridges. However, Western blotting demonstrates the presence of only small amounts of the pancreatic enzymes in water buffalo semen, suggesting that also in this species the seminal-type sequence is not expressed. But it is still possible that the gene is expressed somewhere else in the body or during development. Reconstruction of seminal-type ribonuclease sequences in ancestors of Bovinae and Bovidae indicates no serious abnormalities in the encoded proteins and leads us to the hypothesis that the ruminant seminal-type ribonuclease gene has not come to expression during most of its evolutionary history, but did not exhibit a high evolutionary rate that is generally observed in pseudogenes.

Amino Acid Sequence↗

PubMed1991

A differentiation stage-specific factor interacts with mouse carbonic anhydrase form I gene and a conserved sequence in mammalian beta-globin genes.

We have identified in mouse erythroleukemic (MEL) cells a novel factor which binds to the 3' flanking region of beta-globin genes. Upon induction, this DNA binding factor disappears as beta-globin gene transcription increases. The factor protects 20-30 base pairs (bp) of a sequence which is tightly conserved in beta-globin genes including chicken, human, mouse and rabbit. A very similar sequence binds the factor in the mouse carbonic anhydrase form I gene, whose transcription diminishes upon induction of MEL cells. The factor, or a closely related form, is detected in nonerythroid cells and binding sites have been detected in other genes, including c-myb and immunoglobulin heavy chain-enhancer. We suggest that this factor could play a role in determining the timing of gene expression in several differentiating cell types.

Animals↗

PubMed1987

A cellular protein binds to a conserved sequence in the adenovirus type 2 enhancer.

A sensitive gel retention assay has been utilized to detect proteins from uninfected Hela nuclei which interact with the adenovirus type 2 enhancer. This assay has been employed to monitor fractionation of nuclear extracts. Three enhancer binding factors were resolved by chromatography on DEAE-Sepharose and one of the factors was further purified by chromatography on heparin-Sepharose. DNase protection experiments have shown that the heparin-Sepharose fraction contains a factor which binds predominantly to the conserved sequence GTGGAAATTT present at position 160 in the adenovirus type 2 genome and found in many viral and cellular enhancers. Protection of this sequence from DNase I digestion was abolished by competition with a synthetic duplex oligonucleotide spanning bases 144-181. This region corresponds to the sequence defined by Hen et al. as possessing enhancer function. Competition experiments indicated that the enhancer binding factor also bound, albeit with reduced affinity, to multiple sites in the Ela upstream region located between positions 192 and 353. Within the sequences which compete are regions with homology to the high affinity site at position 160. The enhancer binding factor also binds with high affinity to sequences within the SV40 enhancer demonstrating that this factor interacts with sequences common to both the adenovirus and SV40 enhancers.

Adenoviruses, Human↗

PubMed1990

Sequence conservation among the glucose transporter from the cyanobacterium Synechocystis sp. PCC 6803 and mammalian glucose transporters.

Synechocystis sp. PCC 6803 is capable of facultative photoheterotrophy with glucose as the sole carbon source. Eight mutants that were unable to take up glucose were transformed with plasmids from pooled gene banks of wild-type Synechocystis DNA prepared in an Escherichia coli vector that does not replicate in Synechocystis. One mutant (EG216) could be complemented with all gene banks to restore ability for photoheterotrophic growth. One of the gene banks was fractionated into single clones and plasmid DNA from each clone used to complement EG216. This yielded a 1.5 kb DNA fragment that was sequenced. It contained one complete open reading frame (gtr) whose putative gene product displayed high sequence conservation with the xylose transporter of E. coli and the mammalian glucose transporters. Further, the isolated gtr gene interrupted in vitro by a kanamycin resistance cassette could be used to construct mutants from wild-type Synechocystis sp. PCC 6803 that lacked a functional glucose transporter, thus confirming the identity of the gtr gene with the glucose transporter gene. This is the first prokaryotic glucose transporter known to share a sequence relationship with mammalian glucose transporters and the first sugar transporter from a cyanobacterium characterized at the sequence level.

Amino Acid Sequence↗

PubMed1999

Determination of 5'-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs.

We determined the untranslated 5'-leader sequence for three different isolates of porcine reproductive and respiratory syndrome virus (PRRSV): pathogenic European- and American-types, as well as an American-type vaccine strain. 5'-leader from European- and American-type PRRSV differed in length (220 and 190 nt, respectively), and exhibited only approximately 50% nucleotide homology. Nevertheless, highly conserved areas were identified in the leader of all 3 PRRSV isolates, which constitute candidate motifs for binding of protein(s) involved in viral replication. These comparative data provide a priori knowledge for mutational identification of virulence determinants in the 5' nontranslated part of the PRRSV genome.

5' Untranslated Regions↗

PubMed1992

The Zymomonas mobilis glf, zwf, edd, and glk genes form an operon: localization of the promoter and identification of a conserved sequence in the regulatory region.

The Zymomonas mobilis genes that encode the glucose-facilitated diffusion transporter (glf), glucose-6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), and glucokinase (glk) are clustered on the genome. The data presented here firmly establish that the glf, zwf, edd, and glk genes form an operon, in that order. The four genes of the operon are cotranscribed on a 6.14-kb mRNA. The site of transcriptional initiation for the polycistronic message was mapped by primer extension and nuclease S1 protection analysis. The glf operon promoter region showed significant homology to other highly expressed Z. mobilis promoters, but not to consensus promoters from other bacteria. The highly expressed Z. mobilis promoter set contains two independent, overlapping, conserved sequences that extend from approximately bp -100 to +15 with respect to the transcriptional start sites. Expression of the glf operon was shown to be subject to carbon source-dependent regulation. The mRNA level was threefold higher in cells grown on fructose than in cells grown on glucose. This increase was not the result of differential mRNA processing when cells were grown on the different carbon sources, nor was it the result of differential transcript stability. Degradation of the 6.14-kb glf operon mRNA was biphasic, with initial half-lives of 11.5 min in fructose-grown cells and 12.0 min in glucose-grown cells. Thus, the higher level of glf operon mRNA in fructose-grown cells is the result of an increased rate of transcription. The importance of increasing glf expression in cells growing on fructose is discussed.

Base Sequence↗

PubMed1997

High-degree sequence conservation in LPA kringle IV-type 2 exons and introns.

In the search for factors contributing to the regulation of the Lp(a) lipoprotein concentration, we have sequenced the kringle IV-type 2 encoding exons 1 and 2 together with the flanking intron sequences of the LPA gene in individuals with different serum concentrations of Lp(a) lipoprotein. The high degree of sequence identity between the kringle IV-type 2 repeats made it possible to analyse all the 3-42 kringles simultaneously by polymerase chain reaction and direct DNA sequencing. The strategy used allowed us to determine approximately 700 bp from each kringle IV-type 2 repeat, resulting in a rapid screen of on average 28,000 bp of the LPA gene from each individual. Comparing these bipartite kringle IV-type 2 repeat sequences from 12 individuals with high and 11 individuals with low Lp(a) lipoprotein level revealed that: 1. no sequence polymorphism could be detected in the exons examined; 2. no sequence polymorphism could be detected in the consensus GT/AG splicing signals of exon/intron junctions; and 3. the proximal intron sequences seemed almost completely conserved in the 76-135 bp analysed. Only one position in the intron sequences exhibited the pattern of a G/A polymorphism. We observed no differences between the group with high and the group with low Lp(a) lipoprotein level. The very high conservation of intron sequences could support the hypothesis that the LPA gene evolved relatively recently. The contradictory finding of a corresponding sequence conservation between the human LPA and the plasminogen gene suggests that an evolutionary pressure has preserved these intron sequences over the last 40-90 million years.

Conserved Sequence↗

PubMed2003

Sequence conservation and antibody cross-recognition of clade B human immunodeficiency virus (HIV) type 1 Tat protein in HIV-1-infected Italians, Ugandans, and South Africans.

We determined immune cross-recognition and the degree of Tat conservation in patients infected by local human immunodeficiency virus (HIV) type 1 strains. The data indicated a similar prevalence of total and epitope-specific anti-Tat IgG in 578 serum samples from HIV-infected Italian (n=302), Ugandan (n=139), and South African (n=137) subjects, using the same B clade Tat protein that is being used in vaccine trials. In particular, anti-Tat antibodies were detected in 13.2%, 10.8%, and 13.9% of HIV-1-infected individuals from Italy, Uganda, and South Africa, respectively. Sequence analysis results indicated a high similarity of Tat from the different circulating viruses with BH-10 Tat, particularly in the 1-58 amino acid region, which contains most of the immunogenic epitopes. These data indicate an effective cross-recognition of a B-clade laboratory strain-derived Tat protein vaccine by individuals infected with different local viruses, owing to the high similarity of Tat epitopes.

AIDS Vaccines↗

PubMed1987

Structural analysis of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA.

The human U3 ribonucleoprotein (RNP) has been analyzed to determine its protein constituents, sites of protein-RNA interaction, and RNA secondary structure. By using anti-U3 RNP antibodies and extracts prepared from HeLa cells labeled in vivo, the RNP was found to contain four nonphosphorylated proteins of 36, 30, 13, and 12.5 kilodaltons and two phosphorylated proteins of 74 and 59 kilodaltons. U3 nucleotides 72-90, 106-121, 154-166, and 190-217 must contain sites that interact with proteins since these regions are immunoprecipitated after treatment of the RNP with RNase A or T1. The secondary structure was probed with specific nucleases and by chemical modification with single-strand-specific reagents that block subsequent reverse transcription. Regions that are single stranded (and therefore potentially able to interact with a substrate RNA) include an evolutionarily conserved sequence at nucleotides 104-112 and nonconserved sequences at nucleotides 65-74, 80-84, and 88-93. Nucleotides 159-168 do not appear to be highly accessible, thus making it unlikely that this U3 sequence base pairs with sequences near the 5.8S rRNA-internal transcribed spacer II junction, as previously proposed. Alternative functions of the U3 RNP are discussed, including the possibility that U3 may participate in a processing event near the 3' end of 28S rRNA.

Animals↗

PubMed1987

Isolation and characterization of a conserved sequence highly expressed in tumors and growing cells.

We isolated a cDNA clone, pAH1005, originating from the 6.8-kilobases mRNA of the rat ascites hepatoma cell line, AH60C. The sequence was highly expressed in various tumor tissues and tumor lines in rats, mice and humans. In rats, the expression of the sequence was also observed in fetal liver of 7 and 10 days of gestation, to some extent in the regenerating liver, yet was sparse in normal adult tissues. Additionally, this expression was induced when adult rat hepatocytes in primary monolayer cultures proliferated by the addition of epidermal growth factor and insulin. Southern blot analysis showed no major gene amplification and rearrangement between DNAs from hepatoma and normal liver of the same species. In addition, in vitro nuclear transcription assay revealed that a genomic clone corresponding to the pAH1005 hybridized to the nascent RNA from isolated nuclei of AH60C but not from those of normal rat liver. All these observations suggest that the expression of the conserved sequence we isolated is closely associated with cell growth and is regulated at the transcriptional level.

Animals↗

PubMed1991

Analysis of the promoter region of napin genes from Brassica napus demonstrates binding of nuclear protein in vitro to a conserved sequence motif.

Napin is a seed storage protein from Brassica napus (rape) that is encoded by a gene family. We have isolated and characterized a novel napin gene, napB. Comparisons of the 5'-upstream region of napB to the promoter regions of previously published napin genes reveal that certain sequence motives are evolutionary conserved and may be implicated in gene regulation. These consensus motives, that overlap with purine/pyrimidine stretches, are TACACAT and CATGCA both of which frequently occur as overlapping, direct repeats. Related or identical sequences are also found in the upstream regions of the homologous genes of Arabidopsis thaliana. One copy of the CATGCA motif occurs in close proximity to the TATA box in all the above genes. In this case it overlaps with an octamer sequence (ATGCAAAT) which is a sequence element common in many eukaryotic promoters and enhancers. The TACACAT sequence, as part of a longer purine/pyrimidine stretch, was found to interact with a protein present in crude nuclear extracts from developing B. napus seeds. Napin genes appear to be methylated to almost equal extents whether present in expressing or non-expressing tissue.

2S Albumins, Plant↗

PubMed2003

3MATRIX and 3MOTIF: a protein structure visualization system for conserved sequence motifs.

Computational methods such as sequence alignment and motif construction are useful in grouping related proteins into families, as well as helping to annotate new proteins of unknown function. These methods identify conserved amino acids in protein sequences, but cannot determine the specific functional or structural roles of conserved amino acids without additional study. In this work, we present 3MATRIX (http://3matrix.stanford.edu) and 3MOTIF (http://3motif.stanford.edu), a web-based sequence motif visualization system that displays sequence motif information in its appropriate three-dimensional (3D) context. This system is flexible in that users can enter sequences, keywords, structures or sequence motifs to generate visualizations. In 3MOTIF, users can search using discrete sequence motifs such as PROSITE patterns, eMOTIFs, or any other regular expression-like motif. Similarly, 3MATRIX accepts an eMATRIX position-specific scoring matrix, or will convert a multiple sequence alignment block into an eMATRIX for visualization. Each query motif is used to search the protein structure database for matches, in which the motif is then visually highlighted in three dimensions. Important properties of motifs such as sequence conservation and solvent accessible surface area are also displayed in the visualizations, using carefully chosen color shading schemes.

Amino Acid Motifs↗

PubMed1997

A sequence conserved in vertebrate Hox gene introns functions as an enhancer regulated by posterior homeotic genes in Drosophila imaginal discs.

The intron of the mouse Hoxa-4 gene acts as a strong homeotic response element in Drosophila melanogaster leg imaginal discs. This activity depends on homeodomain binding sites present within a 30 bp conserved element, HB1, in the intron. A similar arrangement of homeodomain binding sites is found in many other potential homeotic target genes. HB1 activity in Drosophila imaginal discs is activated by Antennapedia and more posterior homeotic genes, but is not activated by more anterior genes. Testing a reporter gene construct with mutated binding sites in mouse embryos shows that HB1 is also active in the expression domains of posterior Hox genes in the mouse neural tube.

Animals↗

PubMed1999

Long-distance RNA-RNA interactions and conserved sequence elements affect potato virus X plus-strand RNA accumulation.

Conserved octanucleotide sequences located upstream of two major potato virus X (PVX) subgenomic RNAs (sgRNAs), as well as elements in the 5' end of the genome, affect accumulation of sgRNA. To determine if complementarity between these sequences is important for PVX RNA accumulation, we analyzed the effects of mutations within these elements and compensatory mutations in a tobacco protoplast system and in plants. Mutations in the 5' nontranslated region (NTR mutants) that reduced complementarity resulted in lower genomic RNA (gRNA) and sgRNA levels, whereas mutations to the octanucleotide elements affected only the corresponding sgRNA levels. However, for both the NTR and octanucleotide mutants, the extent of reductions in RNA levels did not directly correlate with the degree of complementarity, suggesting that the sequences of these elements are also important. Mutants containing changes in the NTR and compensatory changes in one of the octanucleotide elements restored levels of gRNA and the other sgRNA species with an unaltered octanucleotide element to those of wild-type. Although compensatory changes significantly increased levels of the sgRNA species with the modified octanucleotide element, levels were not restored to those of wild-type. Our data indicate that long distance RNA-RNA interactions and the sequences of the interacting elements are required for PVX plus-strand RNA accumulation.

5' Untranslated Regions↗