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PubMed1984

Nucleotide sequence of the melB gene and characteristics of deduced amino acid sequence of the melibiose carrier in Escherichia coli.

The nucleotide sequence of the melB gene coding for the melibiose carrier in Escherichia coli has been determined. The melibiose carrier is predicted to consist of 469 amino acid residues, resulting in a protein with a molecular weight of 52,029. The predicted carrier protein is highly hydrophobic (70% nonpolar amino acid residues). The hydropathic profile suggests that there are 10 long hydrophobic segments in the primary structure of the carrier protein. Most of them seem to traverse the membrane. Although the hydropathic profile of the melibiose carrier is similar to that of the lactose carrier as a whole, homology in the primary structure between the two carriers is very low. Furthermore, no homology in the nucleotide sequence is found in the structural genes for the two carriers. However, the nucleotide sequences of the intergenic regions are very similar between the melibiose operon and the lactose operon. There is a typical intercistronic regulatory sequence in the 3'-flanking region of the melB as well as in that of the lacY, which suggests the presence of another gene downstream of the melB.

Amino Acid Sequence↗
PubMed1995

Sequence specificity in the higher-order interaction of the Rev protein of HIV-1 with its target sequence, the RRE.

The Rev protein of human immunodeficiency virus type 1 (HIV-1) multimerizes along RNAs containing the Rev target sequence, the RRE. Although sequence-specific information is recognized in the high affinity or initial interaction, it is not known what role RNA-contained information plays in higher-order binding events. We have quantitatively studied the binding of Rev protein to the primary Rev binding domain (II + III) of wild-type and mutant RREs. RRE mutations that retain the basic secondary structure of wild type can separately and differentially alter the Kds for formation of the first, second, and third Rev/RRE complexes (C1, C2, and C3). The data suggest that Rev recognizes sequence-specific information in the RRE when it forms higher-order complexes. However, the formation of higher-order complexes is not as dependent on sequence-specific information as the first or lowest order binding interaction, which involves recognition of the high-affinity site.

Base Sequence↗
PubMed1996

Arginine 75 in the pseudosubstrate sequence of type Ibeta cGMP-dependent protein kinase is critical for autoinhibition, although autophosphorylated serine 63 is outside this sequence.

Autoinhibitory domains in many protein kinases include either a phosphorylatable substrate-like sequence or a pseudosubstrate sequence. This study shows that Ibeta cGMP-dependent protein kinase (cGK) autophosphorylates Ser-63, which is in an atypical cGK substrate sequence (-59AQKQAS-) that is amino-terminal to the pseudosubstrate motif (-74KRQAI-). cGMP increases the rate of autophosphorylation (approximately 0.8 phosphate/cGK monomer) approximately 3-fold. Autophosphorylation is an intramolecular process since it is independent of cGK concentration. cGMP activation of cGK enhances proteolysis within and near the pseudosubstrate site; treatment of dimeric cGK with three proteases produces three cGK monomers (approximately 67-70 kDa each). Their amino-terminal sequences are 75RQAISAEPT-, 76QAISAEPTAF-, and 86DIQDLSXV-, respectively. cGMP stimulates these kinases by 10-, 2.5-, and 1.4-fold, respectively, compared with a 10-fold effect on intact cGK. Increased basal activity accounts for the diminished stimulation. Thus, the primary autophosphorylation site of Ibeta cGK is well outside the pseudosubstrate site, but Arg-75 in the pseudosubstrate site is critical for autoinhibition. Autoinhibition also involves elements that are carboxyl-terminal to Arg-75.

Amino Acid Sequence↗
PubMed1997

Prediction of enzyme classification from protein sequence without the use of sequence similarity.

We describe a novel approach for predicting the function of a protein from its amino-acid sequence. Given features that can be computed from the amino-acid sequence in a straightforward fashion (such as pI, molecular weight, and amino-acid composition), the technique allows us to answer questions such as: Is the protein an enzyme? If so, in which Enzyme Commission (EC) class does it belong? Our approach uses machine learning (ML) techniques to induce classifiers that predict the EC class of an enzyme from features extracted from its primary sequence. We report on a variety of experiments in which we explored the use of three different ML techniques in conjunction with training datasets derived from PDB and from Swiss-Prot. We also explored the use of several different feature sets. Our method is able to predict the first EC number of an enzyme with 74% accuracy (thereby assigning the enzyme to one of six broad categories of enzyme function), and to predict the second EC number of an enzyme with 68% accuracy (thereby assigning the enzyme to one of 57 subcategories of enzyme function). This technique could be a valuable complement to sequence-similarity searches and to pathway-analysis methods.

Algorithms↗
PubMed1997

The native sequence determines sidechain packing in a protein, but does optimal sidechain packing determine the native sequence?

Globular proteins have highly compact structures and the corresponding packing interactions are widely considered as the principal determinant of the native structure. It is therefore important that theoretical approaches to protein design explicitly take in account packing, which requires that a full atomic representation of the designed protein is maintained. As a first step towards this goal, we have developed in this report an inverse folding algorithm with the aim of specifically designing amino acid sequences which optimise sidechain packing for a given protein fold. The design is performed by a global Monte Carlo optimisation in sequence space, with constant amino acid composition and a full-atom representation of the various protein models. Packing is defined by a Lennard-Jones potential. The program was tested by designing stable sequence variants for the chymotrypsin inhibitor fold. The final protein models showed an increase in intramolecular atomic contacts and a decrease in the overall volume compared to the native structure. Starting from the backbone only of the target structure, the algorithm did gradually retrieve reliable though limited sequence information. Higher compatibility might be achieved by improving the potential, however our results suggest that packing interactions are an essential element of a yet-to-be-defined successful energy function for protein design.

Algorithms↗
PubMed1998

Cloning and sequencing of the bovine flavocytochrome b subunit proteins, gp91-phox and p22-phox: comparison with other known flavocytochrome b sequences.

Neutrophils play an essential role in the cellular defense of the bovine mammary gland and compromised leukocyte function has been linked to the development of bovine mastitis. During mastitis, large numbers of leukocytes migrate into the mammary tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide anion-generating complex known as the NADPH oxidase. The key membrane-associated component of the NADPH oxidase is flavocytochrome b, which is a heterodimer of p22-phox and gp91-phox. Currently, only the human, porcine, murine, and rattus p22-phox and the human, porcine, and murine gp91-phox gene sequences are known. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and analyze expression of bovine flavocytochrome b. Using polymerase chain reaction cloning techniques and a bovine spleen cDNA library we have cloned both of the bovine flavocytochrome b subunits, p22-phox and gp91-phox. Comparison of the bovine sequences with those of other species also revealed important information regarding key structural features of gp91-phox and p22-phox, including location of putative glycosylation sites. This study greatly contributes to our understanding of the potential functional sites of the flavocytochrome b subunits as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases such as mastitis.

Amino Acid Sequence↗
PubMed2004

Determining sequences and post-translational modifications of novel conotoxins in Conus victoriae using cDNA sequencing and mass spectrometry.

A combination of cDNA cloning and detailed mass spectrometric analyses was employed to identify novel conotoxins from Conus victoriae. Eleven conotoxin sequences were determined using molecular methods: one belonging to the A superfamily (Vc1.1), six belonging to the O superfamily (Vc6.1-Vc6.6) and four members of the T superfamily (Vc5.1-Vc5.4). In order to verify the sequences and identify the post-translational modifications (excluding the disulfide connectivity) of three Conus victoriae conotoxins, vc1a, vc5a and vc6a, deduced from sequences Vc1.1, Vc5.1, and Vc6.1, respectively, liquid chromatography/electrospray ionization ion trap mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanospray ionization ion trap mass spectrometry with collisionally induced dissociation were performed on reduced and alkylated venom fractions. We report that vc1a, the native form of alpha-conotoxin Vc1.1 (an unmodified 16 amino acid residue peptide that has notable pain-relieving capabilities), includes a hydroxyproline and a gamma-carboxyglutamate residue. Conotoxin vc5a is a 10-residue peptide with two disulfide bonds and a hydroxyproline and vc6a is a 25 amino acid peptide with three disulfide bonds.

Amino Acid Sequence↗
PubMed1993

Complete sequence of a bovine alpha-lactalbumin pseudogene: the region homologous to the gene is flanked by two directly repeated LINE sequences.

A 3-kb fragment hybridizing with a bovine alpha-lactalbumin cDNA probe was isolated from a bovine genomic library and completely sequenced. An internal fragment beginning downstream from exon 2, as already reported for another pseudogene, but ending in the 3'-untranslated region of exon 4 shares 78% sequence similarity with the bovine alpha-lactalbumin gene. This region is flanked by two directly repeated LINE sequences. The 5' ends of the fragment and of the aforementioned pseudogene share a specific nucleotide stretch, which suggests that they might have had a common origin.

ATP Synthetase Complexes↗
PubMed1996

Systematic sequencing of the human HLA-A/HLA-F region: establishment of a cosmid contig and identification of a new gene cluster within 37 kb of sequence.

The class I region of the human histocompatibility complex is characterized by a high density of genes and pseudogenes and a complex structural organization. To elucidate the complete structure of the HLA-A/HLA-F region with a view to defining its contents in genes and pseudogenes, we developed a strategy of systematic sequencing. This report describes the establishment of a cosmid contig spanning most of the region and the analysis of a 37-kb sequence from one of the cosmids. Four new genes, organized with the HCG-V gene in a clustered structure, have been identified. Two of these contain a zinc finger motif characteristic of DNA-binding proteins. The former, a member of the C3HC4 protein family, is highly expressed in prostate and contains a B30-2-like sequence identified in several genes mapped within the class I region. The latter, which is ubiquitously expressed, is the human equivalent of the yeast polymerase IA12.2 subunit and of the murine tctex6 gene. Of the two other genes, one remains an anonymous gene with no particular feature, while the fourth, specifically expressed in testis, is the human equivalent of the murine tctex4 gene. This cluster, located in a region corresponding to a syntenic unit between mouse and human, appears to be highly conserved.

Amino Acid Sequence↗
PubMed1998

The genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins.

Sequencing and computer analysis of the left (52,283 bp) and right (49,649 bp) variable DNA regions of the cowpox virus strain GRI-90 (CPV-GRI) has revealed 51 and 37 potential open reading frames (ORFs), respectively. Comparison of the structure-function organization of these DNA regions of CPV-GRI with those previously published for corresponding regions of genomes of vaccinia virus, strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR); and variola major virus, strains India-1967 (VAR-IND), Bangladesh-1975 (VAR-BSH); and alastrim variola minor virus, strain Garcia-1966 (VAR-GAR), was performed. Within the left terminal region under study, an extended DNA sequence (14,171 bp), unique to CPV, has been found. Within the right region of the CPV-GRI genome two segments, which are unique to CPV DNA (1579 and 3585 bp) have been found. Numerous differences have been revealed in the genetic structure of CPV-GRI DNA regions, homologous to fragments of the genomes of the above-mentioned orthopoxvirus strains. A cluster of ORFs with structural similarity ot immunomodulatory and host range function of other poxviruses have also been detected. A comparison of the sequences of ORF B, crmA, crmB, crmC, IMP, and CHO hr genes of CPV Brighton strain (CPV-BRI) with the corresponding genes in strain GRI-90 have revealed an identity at the amino acid level ranging from 82 to 96% between the two strains. The findings are significant in light of the recent demonstration of CPV as an important poxvirus model system to probe the precise in vivo role(s) of the unique virally encoded immunomodulatory proteins. Also, the presence of a complete and intact repertoire of immunomodulatory proteins, ring canal proteins family, and host range genes indicates that CPV may have been the most ancient of all studied orthopoxviruses.

Amino Acid Sequence↗
PubMed1995

Isolation, sequencing and expression of cDNA sequences encoding uroporphyrinogen decarboxylase from tobacco and barley.

We have cloned and sequenced a full-length cDNA for uroporphyrinogen decarboxylase (UROD, EC 4.1.1.37) from tobacco (Nicotiana tabacum L.) and a partial cDNA clone from barley (Hordeum vulgare L.). The cDNA of tobacco encodes a protein of 43 kDa, which has 33% overall similarity to UROD sequences determined from other organisms. We propose that tobacco UROD has an N-terminal extension of 39 amino acid residues. This extension is most likely a chloroplast transit sequence. The in vitro translation product of UROD was imported into pea chloroplasts and processed to ca. 39 kDa. A truncated cDNA, from which the putative transit peptide had been deleted, was used to over-express the mature UROD in Escherichia coli. Purified protein showed UROD activity, thus providing an adequate source for subsequent enzymatic characterization and inhibition studies. Expression of UROD was investigated by northern and western blot analysis during greening of etiolated barley seedlings, and in segments of barley primary leaves grown under day/night cycles. The amount of RNA and protein increased during illumination. Maximum UROD-RNA levels were detected in the basal segments relative to the top of the leaf.

Amino Acid Sequence↗
PubMed2005

Completion of the sequence of a cetacean morbillivirus and comparative analysis of the complete genome sequences of four morbilliviruses.

The gene encoding the large (L) protein and the genome termini of the dolphin strain of cetacean morbillivirus (CeMV) were sequenced. The CeMV genome is 15702 nucleotides long and has been compared with other available morbillivirus genome sequences in regards to the "rule of six" and the "phase" of any particular nucleotide, defined as its position within a given hexamer, which here is defined as a group of six nucleotides starting from the 3' end of the genomic RNA. With exception of the position of the start of the F gene, the phase of the transcription start sites of each gene is strictly conserved between the morbilliviruses, but each gene is in a different phase. The lengths of gene transcripts differ between viruses by multiples of six nucleotides with exception of the M and F transcripts. The differences between the various morbilliviruses result from deletions or insertions of multiples of six nucleotides in the 3' and 5' UTRs of the different viral genes. The four bases were distributed non-randomly over the six positions in the hexamer boxes. However, the distribution patterns of each of the four bases indicated that multiples of three were more prevalent than those of six nucleotides. This reflected the positions of nucleotides in codons and codon usage in the reading frames. The L protein of CeMV was found to be 2183 amino acids in length and similar to that of MV and RPV. The CeMV L protein sequence was found to be equidistant between those of the CDV/PDV and MV/RPV subgroups of the morbilliviruses. This concurs with the analyses carried out on the other structural proteins.

3' Untranslated Regions↗
PubMed1990

Cloning and sequence of IS1000, a putative insertion sequence from Thermus thermophilus HB8.

The complete nucleotide sequences of two copies of a putative insertion sequence IS1000 from Thermus thermophilus HB8 are presented. IS1000 is 1196 base pairs long, contains a long open reading frame which could code for a protein of 317 amino acids, and has imperfect terminal inverted repeats of 6 base pairs (confirmed by the terminal sequencing of 4.5 copies of IS1000), but does not cause a target site duplication. There are at least 6 copies of IS1000 in the genome of T. thermophilus HB8. A search of the GEN-EMBL data base revealed that the putative 317 amino acid protein had significant homology with open reading frames in the transposable elements IS110 of Streptomyces coelicolor and IS492 of Pseudomonas atlantica.

Amino Acid Sequence↗
PubMed1995

Irregular polymerase chain reaction-sequence-specific oligonucleotide hybridization patterns reveal seven new HLA-DRB1 alleles related to DR2, DR3, DR6, DR8, and DR11. Implications for sequence-specific priming.

In the past 3 years we have typed over 7000 individuals for HLA-DRB using a nonradioactive PCR-SSO method. The use of locally developed computer programs simplified data input and the interpretation of the DRB PCR-SSO readings. In this way we detected a number of samples with unexpected hybridization patterns. DRB1 exon 2 segments of these samples were amplified, cloned, and sequenced and appeared to identify seven new DRB alleles: DRB1*0304, a DRB1*0301 variant, was observed in three unrelated Caucasoid individuals; DRB1*1606, which is very similar to *1603; DRB1*1113 is a *1101 variant with some *1401 sequences; DRB1*1310 is *1301-like; DRB1*1311 is similar to *1305 and *1307; DRB1*1416 is a *1401 sequence with a HV3 derived from *1301; DRB1*0808 was found in an Ethiopian individual. Next, we studied the effectiveness of PCR-SSP typing of the newly defined DRB1 alleles. Only two variants were distinguished as odd by PCR-SSP and two were typed as regular specificities. Three alleles were not amplified by the primer sets used. As compared to PCR-SSO, the PCR-SSP typing method using currently available typing kits clearly has limitations as far as the recognition of new and variant alleles is concerned. The products of some of these new alleles may be distinguished using conventional serology.

Alleles↗
PubMed1996

Sequence specific binding of cytosolic proteins to a 12 nucleotide sequence in the 5' untranslated region of FMR1 mRNA.

The 5' untranslated region of human FMR1 mRNA is highly conserved, including a 26 nucleotide sequence containing a tandem 12 nucleotide repeat of (G/C)CU(C/G)CCGG(G/A)G(G/C)(G/C) which predates the evolutionary divergence between birds and mammals. We show here that this 12 nucleotide sequence in FMR1 mRNA is a specific binding site for small (< 20 kDa) cytosolic proteins of rat brain. Point mutation analysis identified two guanine residues in this 12 nucleotide repeat which are essential for protein binding. The 12 nucleotide motif sequence was found in the 5'UTR of at least 15 other genes and could be a common target site for these cytosolic RNA-binding proteins.

Animals↗
PubMed2000

Sequence diversity of intervening sequences (IVSs) in the 23S ribosomal RNA in Salmonella spp.

Intervening sequences (IVSs) occur sporadically in the rrl (ribosomal RNA large) genes for 23S ribosomal RNA (rRNA) at helix-25 (base pair 550) and helix 45 (base pair 1170) in several bacterial genera, including Salmonella, Yersinia, Proteus, and Providencia, representing the Enterobacteriaceae, but are missing from other genera such as Escherichia. These sequences are transcribed, but later excised without re-ligation during RNaseIII processing of the rRNA, resulting in fragmented 23S rRNA. The IVSs from 22 strains of the SARB (Salmonella Reference Collection B) set were amplified by PCR and sequenced.IVSs with 90% or more sequence identity were placed in the same family; Salmonella has three families of IVSs in helix-25 (A, B, and C) and two in helix-45 (M and O). The rRNA secondary structure for the IVSs predicted from the mfold program reveals a primary stem of about 14bp, which is the postulated RNaseIII cleavage site, and a secondary region of stems and loops. The primary stem is considerably well conserved, with a high rate of compensatory mutations (positional covariants), confirming the reality of the secondary structure and indicating that removal of the IVSs exerts a positive selective pressure to retain the secondary structure. The pattern of possession and presence of families of IVSs was diverse and could not be related to the proposed ancestry of the strains as revealed by the multi-locus enzyme electrophoresis pattern of the strains, suggesting that the IVSs are transferred between strains by lateral transfer. Helix-25 IVSs from families A, B, and C of Salmonella and D of Proteus, which share almost identical primary stems, are placed in superfamily I, while the primary stems of other IVSs from Proteus and Providencia are unrelated to superfamily I and are thus placed into superfamily II; this indicates lateral transfer of members of superfamily I between Proteus and Salmonella, but an independent origin of IVSs of superfamily II in Proteus and Providencia.

Base Sequence↗
PubMed1999

Complete nucleotide sequence of bean pod mottle virus RNA1: sequence comparisons and evolutionary relationships to other comoviruses.

The complete nucleotide sequence of bean pod mottle comovirus (BPMV) RNA 1 was determined. It is 5983 nucleotides long, excluding the poly(A) tail, and encodes a polyprotein of 1850 amino acid (aa) residues. Multiple alignments of the deduced aa sequence of BPMV polyprotein with those of cowpea mosaic virus (CPMV), red clover mottle virus (RCMV) and cowpea severe mosaic virus (CPSMV) indicated that BPMV RNA1 encodes the predicted set of five mature proteins: the equivalent of CPMV 32K protease cofactor, 58K putative helicase, VPg, 24K protease and 87K putative RNA-dependent RNA polymerase. Of the four proposed cleavage sites in BPMV RNA1 polyprotein, the one at the 32K/58K site (Q/A) is distinct for BPMV polyprotein and those at the 58K/VPg and VPg/24K junctions (Q/S and Q/M, respectively) are identical in all four comovirus polyproteins. Sequence comparison and phylogenetic analysis revealed that BPMV RNA1 is more closely related to CPSMV than to CPMV or to RCMV.

Amino Acid Sequence↗
PubMedDate not supplied

Expressed sequence tags for the chicken genome from a normalized 10-day-old White Leghorn whole embryo cDNA library: 1. DNA sequence characterization and linkage analysis.

Expressed sequence tags (ESTs) provide a rapid and reliable method for gene discovery as well as a resource for the large-scale analysis of gene expression of known and unknown genes. Here we describe a normalized cDNA library developed from a 10-day-old White Leghorn chicken whole embryo. The utility of the library was evaluated by partial sequencing of 99 randomly selected insert-containing clones and the analysis of EST-targeted genomic regions for single nucleotide polymorphisms (SNPs) in the East Lansing chicken reference DNA mapping panel. Using stringent match criteria of percent identity of 80 or higher across a length of 50 or more bases, 46 ESTs matched database sequences including previously reported Gallus gallus genes. Thirty-seven of the 50 primer pairs developed from 50 unique ESTs amplified a single fragment. The size of the 37 amplicons ranged from 276 to 693 bp for a total of 17,508 and an average of 473. About 70% of the SNPs detected were either G-->A or C-->T transition. The number of SNPs detected within the amplicons from EST-targeted genomic regions ranged from 0 to 4 for a total of 65 and a frequency of about 1 every 470 bases. About 35% of the amplicons contained only 1 SNP, while 19% had 4 SNPs. Using the SNPs that were informative in the East Lansing reference panel, 17 ESTs were mapped on the East Lansing chicken genetic map. The ESTs described, as well as the nucleotide variants identified within the EST-targeted genomic regions, represent significant resources for genome analysis in the chicken.

Animals↗