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PubMed1976

A novel manual method for protein-sequence analysis.

A novel manual method for protein-sequence analysis is described. Three peptides, the hexapeptide (Leu-TRP-Met-Arg-Phe-Ala), insulin A chain and glucagon were used to test this technique. Peptides (1 or 2 nmol) were hydrolysed with acid and their qualitative amino acid compositions were confirmed by reacting with 4-NN-dimethylaminoazobenzene-4'-sulphonylchloride and 4-NN-dimethylaminoazobenzene 4'-isothiocyanate. Sequence determination of 20-200 nmol of peptide was then performed by the combined use of phenyl isothiocyanate and 4-NN-dimethylaminoazobenzene 4'-isothiocyanate, a new procedure that is analogous to the dansyl-Edman method with the replacement of dansyl chloride by 4-NN-dimethylaminoazobenzene 4'-isothiocyanate as the N-terminal residue determination reagent. On t.l.c. this new N-terminal reagent gave brightly coloured 4-NN-dimethylaminoazobenzene-4-thiohydantoins of amino acids and showed the following advantages: (1) the detection sensitivity is in the pmol range; (2) u.v. observation is not required; (3) there is no destruction of acid-labile amino acids; (4) two-dimensional t.l.c. separation is adequate to identify 24 amino acids, except leucine and isoleucine (this pair of amino acids can be resolved by using 4-NN-dimethylaminoazobenzene-4'-sulphonyl chloride); (5) the determination of a new N-terminal residue (from coupling to t.l.c. identification) takes only 3 h; (6) the colour difference beteen isothiocyanate, thiocarbamoyl and thiohydantoin derivatives facilitates the identifications.

Amino Acid Sequence↗

PubMed1996

Primary structures and sequence analysis of human ribosomal proteins L39 and S27.

We report here the primary structures and sequence analysis of the human ribosomal protein L39 (hRPL39) and S27 (hRPS27). The hRPL39 cDNA is 352 bp in size encoding a predicted protein of 51 amino acids. The region KRRHWRRTKL near the carboxyl end is conserved between human, rat, maize, C. elegans and yeast. The hRPS27 cDNA is 321 bp in size encoding a deduced protein of 84 amino acids. When the deduced amino acid sequence of hRPS27 was compared with that of rat ribosomal protein S27 and human metalloproteinstimulin-1 (MPS-1), identity levels of 96.4% and 100% were obtained respectively. A potential polyadenylation signal AACAAA is found in the MPS-1 cDNA but the more frequently used AATAAA sequence is present in the hRPS27 cDNA. The carboxyl-terminal cysteine arrangement in hRPS27 is similar to the family of C4 zinc finger DNA-binding proteins.

Amino Acid Sequence↗

PubMed2001

Sequence analysis of feline caliciviruses isolated from the oral cavity of clinically normal domestic cats (Felis catus) in Florida.

Four isolates (7.3 per cent) of feline calicivirus (FCV), from oropharyngeal swabs taken from 55 unvaccinated apparently healthy cats, were identified by electron microscopy and reverse transcription-polymerase chain reaction (RT-PCR). A 671-bp fragment, comprising part of region B, all of regions C, D, and E and part of region F of the FCV capsid protein gene, was amplified from each isolate by RT-PCR and cloned for sequence analysis. Amino acid sequence comparison of these regions revealed significant sequence divergence from the F9 vaccine strain within regions C, E, and F. The hypervariable region E of the four Florida isolates and the NADC isolate contained three fewer amino acids than the commonly used F9 vaccine strain. This work provides support to the idea that currently circulating FCV strains may differ substantially from presently used vaccine strains.

Amino Acid Sequence↗

PubMed2006

Multilocus sequence analysis of Penicillium and Eupenicillium species.

Taxonomy of Hyphomycetes has always been a challenging problem, with experts viewing species in different ways and modifying the taxonomy of groups to reflect their best evaluation of species limits and concepts. The advent of phylogenetic analysis, relatively easy DNA sequencing techniques and PCR has provided an opportunity for mycology to move from a strictly morphological analysis of species to phylogenetic analysis of DNA sequences. Phylogenetic theory dictates that data from different loci will produce congruent or at least non-contradictory evolutionary histories of a clonal lineage. Tests of tree congruence such as the index of association can show whether lineages are clonal, and has revealed that some species long thought to be clonal are cryptically recombining. Genealogical concordance phylogenetic species recognition allows unambiguous identification of species boundaries.

Artifacts↗

PubMed1981

Inactivation of the Serratia marcescens gene for the lipoprotein in Escherichia coli by insertion sequences, IS1 and IS5; sequence analysis of junction points.

A pBR322-derived plasmid pKEN221 carrying a Serratia marcescens lpp gene overproduces the outer membrane lipoprotein in an Escherichia coli lpp- cell. However, when this strain was continuously cultured in a rich medium for about thirty generations, many Lpp- mutants were accumulated. Out of six mutants analyzed, three were found to carry insertion mutation in the lpp gene in pKEN221. From resistance enzyme mapping and hybridization analysis of the mutant plasmid DNA, it was found that two mutants were caused by insertion sequence IS1 and one by IS5. Nucleotide sequence analysis of these mutant DNAs revealed that both IS1 and IS5 insertions occurred in the A-T rich 5' untranslated region of the lpp gene. While the IS1 insertion resulted in a direct duplication of a nine-base-pair sequence in the original pKEN221 DNA at the junction with IS1, the IS5 insertion resulted in a direct duplication of a four-base-pair sequence. IS5 was found to contain inverted-repeat sequences of twelve nucleotides at its exact ends. This is the first example of the nucleotide sequence analysis of an IS5 insertion mutation. By Southern blot hybridization, the E. coli chromosomal DNA was found to contain about ten copies of IS5.

Bacterial Proteins↗

PubMed2004

Clinical evaluation and sequence analysis of the complete mitochondrial genome of three Chinese patients with hearing impairment associated with the 12S rRNA T1095C mutation.

Mutations in mitochondrial DNA (mtDNA), particularly those in the 12S rRNA gene, have been shown to be associated with sensorineural hearing loss. Here we report the clinical and sequence analysis of the entire mitochondrial genome in three Chinese subjects with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluation showed a variable phenotype of hearing impairment including the age of onset and audiometric configuration in these subjects. Sequence analysis of the complete mitochondrial genomes in three subjects showed the distinct sets of mtDNA polymorphism, in addition to the identical mitochondrial 12S rRNA T1095C mutation. This mutation was previously identified to be associated with hearing impairment in three families from different genetic backgrounds. The T1095C mutation was absent in 364 Chinese control. In fact, the occurrence of the T1095C mutation in these several genetically unrelated subjects affected by hearing impairment strongly indicates that this mutation is involved in the pathogenesis of hearing impairment. Among other nucleotide changes, the A2238G and T2885C mutations in the 16S rRNA, the I175V mutation in the CO2, the F16L mutation in the A6 and the V112M mutation in the ND6 exhibited a high evolutionary conservation. These data suggest that the T1095C mutation may be associated with aminoglycoside-induced and non-syndromic hearing impairments and A2238G and T2885C mutations in the 16S rRNA, the I175V mutation in the CO2, the F16L mutation in the A6 and the V112M mutation in the ND6 may contribute to the phenotypic expression of the T1095C mutation in these subjects.

Asian People↗

PubMed2006

Space-time image sequence analysis: object tunnels and occlusion volumes.

We address the issue of image sequence analysis jointly in space and time. While typical approaches to such an analysis consider two image frames at a time, we propose to perform this analysis jointly over multiple frames. We concentrate on spatiotemporal segmentation of image sequences and on analysis of occlusion effects therein. The segmentation process is three-dimensional (3-D); we search for a volume carved out by each moving object in the image sequence domain, or "object tunnel," a new space-time concept. We pose the problem in variational framework by using only motion information (no intensity edges). The resulting formulation can be viewed as volume competition, a 3-D generalization of region competition. We parameterize the unknown surface to be estimated, but rather than using an active-surface approach, we embed it into a higher dimensional function and apply the level-set methodology. We first develop simple models for the detection of moving objects over static background; no motion models are needed. Then, in order to improve segmentation accuracy, we incorporate motion models for objects and background. We further extend the method by including explicit models for occluded and newly exposed areas that lead to "occlusion volumes," another new space-time concept. Since, in this case, multiple volumes are sought, we apply a multiphase variant of the level-set method. We present various experimental results for synthetic and natural image sequences.

Algorithms↗

PubMed1984

A comprehensive sequence analysis program for the IBM personal computer.

We have developed a versatile program for the analysis of nucleic acid and protein sequences on the IBM Personal Computer. The program is interactive and self-instructing. It contains all the features generally found in sequence analysis programs on large computers, including extensive homology routines, as well as new procedures for the entry of sequence data. The program contains facilities to store and utilize the entire Nucleic Acid Sequence Data Bank. We have devised a new algorithm to find restriction enzyme sites, which allows our microcomputer program to find all sites on a small plasmid for 100 different enzymes in 1 to 2 minutes.

Amino Acid Sequence↗

PubMed1976

Purification and sequence analysis of the mRNA coding for an immunoglobulin heavy chain.

A mutant cell line (IF2) derived from the mouse myeloma MOPC 21 has been used for the isolation and sequence analysis of H-chain mRNA. The IF2 cells synthesise an H-chain of reduced size in which the CH1 homology region is missing. Sizing of the IF2 H-chain mRNA and wild-type H-chain mRNA revealed that the deletion is expressed at the mRNA level. The mutant H-chain mRNA sedimented at 16-S, enabling effective resolution from 18-S ribosomal RNA. In experiments using IF2 cells labelled with [32P]phosphate, the 16-S mRNA was purified by oligo(T)-cellulose chromatography. Polyacrylamide gel analysis of the poly(A)-containing fraction showed the presence of a single radioactive band. Comparison of the mobility of this band relative to markers of known molecular weight revealed that the molecule contained about 1600 nucleotides. Digestion of the 32-P-labelled mRNA with T1 ribonuclease and two-dimensional fractionation of the resulting oligonucleotides yielded a 'finger-print' suitable for a preliminary sequence analysis. By using the established amino acid sequence of the IF2 H-chain and a knowledge of the genetic code, 14 oligonucleotides were assigned within the constant region and four within the variable region of the IF2 H-chain. This sequence data accounts for 19.5% of the coding region. Several other oligonucleotides, which could not be assigned within the coding region but which occurred in approximately molar yield, have also been partially characterised. These oligonucleotides are presumably derived from the untranslated regions of mRNA.

Amino Acid Sequence↗

PubMed2001

Sequence analysis of a 101-kilobase plasmid required for agar degradation by a Microscilla isolate.

An agar-degrading marine bacterium identified as a Microscilla species was isolated from coastal California marine sediment. This organism harbored a single 101-kb circular DNA plasmid designated pSD15. The complete nucleotide sequence of pSD15 was obtained, and sequence analysis indicated a number of genes putatively encoding a variety of enzymes involved in polysaccharide utilization. The most striking feature was the occurrence of five putative agarase genes. Loss of the plasmid, which occurred at a surprisingly high frequency, was associated with loss of agarase activity, supporting the sequence analysis results.

Agar↗

PubMed1976

Nucleotide clusters in deoxyribonucleic acids. XIII. Sequence analysis of the longer unique pyrimidine oligonucleotides of bacteriophage S13 DNA by a method using unlabeled atarting oligonucleotides.

A method has been designed for sequence analysis of unlabeled oligodeoxynucleotides of chain length up to 20 nucleotides with no restriction on base composition. The unlabeled oligonucleotide preparation, is partially degraded with spleen exonuclease to give a series of products each differing in size by one nucleotide. The oligonucleotides in the digest are 5'-32 P terminally labeled with [psi-32] P ATP and T4 polynucleotide kinase, the excess ATP removed by chromatography on Sephadex G-25 then the oligonucleotides fractionated according to change length on DEAE-Sephadex. Each isostich fraction is analyzed for base composition and the nucleotide at the 5' terminus determined by its 32P label, resulting in direct read off of the sequence up to the penultimate 3'- terminal nucleotide. The 3'-terminal dinucleotide is analyzed by DEAE-cellulose chromatography of the Sephadex G-25 dinucleotide fraction. The method has been demonstrated by sequence analysis of the unique longer pyrimidine oligonucleotides C5T6, C2T8, C6T4 and C6T3 from S13 DNA. The sequences have extensive internal sequence homology.

Base Sequence↗

PubMed1983

Sequence analysis of rat hypothalamic corticotropin-releasing factor with the o-phthalaldehyde strategy.

Sequence analysis was performed on a 41-residue polypeptide that has been identified as the predominant form of high intrinsic corticotropin-releasing activity of rat hypothalamus. The sequence of residues 1-39 of this corticotropin-releasing factor (CRF) was determined by Edman degradation of a partially purified peptide in a highly sensitive spinning cup sequencer after selective blocking of CRF or its main contaminant with o-phthalaldehyde. This approach was validated by peptide mapping of CRF of a highly purified preparation. Peptide mapping was accomplished with reverse-phase high-pressure liquid chromatography of CRF fragments obtained by digestion with clostripain. The identities of the fragments cleaved from CRF were established by chromatographic comparison with synthetic peptides, amino acid analysis, and Edman degradation. On the basis of these experiments, the primary structure of rat hypothalamic CRF was established to be H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn - Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. It is expected that the o-phthalaldehyde strategy will facilitate the sequence analysis of partially purified peptides containing proline residues.

Amino Acid Sequence↗

PubMed1997

C-terminal sequence analysis of peptides and proteins using carboxypeptidases and mass spectrometry after derivatization of Lys and Cys residues.

C-Terminal sequence analysis of peptides and proteins using carboxypeptidase digestion in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is convenient for protein and peptide characterization. After a short digestion, a sequence up to 20 residues can be identified, but the total number depends on the individual sequence. Due to the accuracy limits of the MALDI time-of-flight arrangement, the assignment of several residues with close mass values, including Lys/Glx, may remain ambiguous. We have used derivatization of lysine residues by guanidination to overcome the problem of Lys identification. The reaction is rapid and specific and results in full derivatization. In the case of Cys-containing peptides, problems arise from the fact that carboxypeptidases Y and P do not cleave peptides that contain nonderivatized cystine, cysteic acid, or (carboxymethyl)cysteine. Successful identification of Cys residues within the sequence is instead achieved by conversion of Cys to 4-thialaminine by (trimethylamino)-ethylation. The two derivatizations of Lys and Cys side chains provide opportunities for proton attachment and therefore facilitate the analysis by MALDI-MS. This C-terminal sequence analysis method is also useful for large proteins after fragmentation with specific enzymes.

Amino Acid Sequence↗

PubMed1993

Determination of the position of the boundaries of the terminal repetitive sequences within the genome of molluscum contagiosum virus type 1 by DNA nucleotide sequence analysis.

The repetitive DNA sequences of the genome of Molluscum contagiosum virus type 1 (MCV-1) have been localized within the terminal regions of the viral genome corresponding to the BamHI MCV-1 DNA fragments B (18 kbp; 0 to 0.095 map units (m.u.)) and E (10.5 kbp; 0.944 to 1 m.u.). The fine mapping of these particular regions of the genome of MCV-1 revealed that the boundaries of the terminal repetitive DNA sequences of the viral genome are located within the DNA sequences of the HindIII MCV-1 DNA fragments K (3.8 kbp; 0.014 to 0.036 m.u.) and J1 (4.1 kbp; 0.962 to 0.985 m.u.). The exact position of the boundary of the repetitive DNA sequences was determined by DNA nucleotide sequencing. The HindIII DNA fragments K and J1 compose 3859 and 4107 bp, respectively. The DNA sequences of HindIII MCV-1 DNA fragment K possess repetitive DNA sequences between the nucleotide positions 1 and 1675 which are homologous to the inverted and complementary DNA sequences of the HindIII MCV-1 DNA fragment J1 between the nucleotide positions 2437 and 4107 (1670 bp). The degree of DNA sequence homology detected between the repetitive DNA sequences in the HindIII DNA fragments K and J1 of the viral genome was found to be 98%. The number of open reading frames (ORFs) detected by the analysis of the DNA sequences of the HindIII MCV-1 DNA fragments K and J1 was found to be 14 (70 to 219 amino acid residues) and 11 (70 to 365 amino acid residues), respectively.

Base Sequence↗

PubMed1998

Sequence analysis of libraries from individual human blastocysts.

PURPOSE: It has recently become possible to construct cDNA libraries from individual human blastocysts to investigate the expression of embryonic genes in human preimplantation development. We have previously reported the expression of beta-actin, CD-59, and homeobox OCT-3 and identified almost-complete homology of sequences to human histone 3.1 and human ribosome protein S25. In the present paper, our further sequencing analysis of cDNA libraries from single human blastocysts is described. METHODS: cDNA libraries were constructed from 13 blastocysts. Sequence analysis was performed in 120 clones from one of these cDNA libraries with fragments of 50 to 1000 bp. Their sequence identity was analyzed using the expressed sequence tag (EST) database. RESULTS: The presence of two housekeeping genes, hexokinase I and serine/threonine phosphorylase, and four other ESTs was demonstrated, the identity of which, with particular gene expression in preimplantation development, has not yet been established. CONCLUSIONS: The data demonstrate the usefulness of constructing cDNA libraries from individual human blastocysts and their values in the analysis of genetic expression in human preimplantation development.

Blastocyst↗

PubMed2001

Hepatitis C virus genotyping based on 5' noncoding sequence analysis (Trugene).

Hepatitis C virus (HCV) genotyping of samples from 184 patients with chronic HCV infection by the Trugene 5'NC genotyping kit, based on sequence analysis of the 5' noncoding region (5' NCR), and the InnoLiPA assay was evaluated. In addition to these methods, the 184 samples were also analyzed by sequencing of part of the NS5B of the HCV genome after in-house PCR amplification, as a means of validating results obtained with the 5' NCR. The distribution of the genotypes typed by NS5B sequence analysis was as follows: 1a, 41 samples; 1b, 58 samples; 1d, 1 sample; 2a, 5 samples; 2b, 2 samples; 2c, 7 samples; 3a, 46 samples; 4a, 7 samples; 4c, 1 samples; 4e, 9 samples; 5a, 6 samples; 6a, 1 sample. The Trugene and InnoLiPA assays gave concordant results within HCV types in 100% of cases. The ability to discriminate at the subtype level was 76 and 74% for the Trugene and the InnoLiPA assays, respectively.

5' Untranslated Regions↗

PubMed2000

Applicability of the alkylation chemistry for chemical C-terminal protein sequence analysis.

We have evaluated the alkylation chemistry first described some years ago by Boyd et al. which is now routinely applied in a commercial instrument. We have found that the low repetitive yields observed during these analyses are due to the formation of a major side product when alkylating the C-terminal thiohydantoin. This side product, resistant to the chemical cleavage methods currently used, was characterized by NMR experiments in solution. We further demonstrate that chemical C-terminal sequence analysis of proteins using the alkylation chemistry is feasable with low picomole amounts of material. High-sensitivity C-terminal sequencing allows a complementary approach by which a protein is first subjected to N-terminal Edman degradation followed by C-terminal sequence analysis, limiting the amount of material necessary for the characterization of the protein under study. This limited C-terminal sequence information is often sufficient to solve problems that cannot be solved by applying any other analytical method commonly used today.

Alkylation↗

PubMed2004

Evolutionary genetic analysis of the emergence of epidemic Vibrio cholerae isolates on the basis of comparative nucleotide sequence analysis and multilocus virulence gene profiles.

Vibrio cholerae, the causative agent of cholera, is a natural inhabitant of the aquatic ecosystem. We examined a unique collection of V. cholerae clinical and environmental isolates of widespread geographic distribution recovered over a 60-year period to determine their evolutionary genetic relationships based on analysis of two housekeeping genes, malate dehydrogenase (mdh) and a chaperonin (groEL). In addition, the phylogenetic distribution of 12 regions associated with virulence was determined. Comparative sequence analysis of mdh revealed that all V. cholerae O1 and O139 serogroup isolates belonged to the same clonal lineage. Single-strand conformational polymorphism (SSCP) analysis of these O1 and O139 strains at groEL confirmed the presence of an epidemic clonal complex. Of the 12 virulence regions examined, only three regions, Vibrio seventh pandemic island 1 (VSP-I), VSP-II, and RS1, were absent from all classical V. cholerae isolates. Most V. cholerae El Tor biotype and O139 serogroup isolates examined encoded all 12 virulence regions assayed. Outside of V. cholerae O1/O139 serogroup isolates, only one strain, VO7, contained VSP-I. Two V. cholerae El Tor isolates, GP155 and 2164-78, lacked both VSP-I and VSP-II, and one El Tor isolate, GP43, lacked VSP-II. Five non-O1/non-O139 serogroup isolates had an mdh sequence identical to that of the epidemic O1 and O139 strains. These isolates, similar to classical strains, lack both VSP-I and VSP-II. Four of the 12 virulence regions examined were found to be present in all isolates: hlyA, pilE, MSHA and RTX. Among non-O1/non-O139 isolates, however, the occurrence of the additional eight regions was considerably lower. The evolutionary relationships and multilocus virulence gene profiles of V. cholerae natural isolates indicate that consecutive pandemic strains arose from a common O1 serogroup progenitor through the successive acquisition of new virulence regions.

Bacterial Proteins↗