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At least 883 records · Page 49Linked to original sources
PubMed2000

The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression.

A gene family of small membrane proteins, represented by phospholemman and the gamma subunit of Na,K-ATPase, was defined and characterized by the analysis of more than 1000 related ESTs (expressed sequence tags). In addition to new and more complete cDNA sequence for known family members (including MAT-8, CHIF, and RIC), the findings included two new family members and new splicing variants. A large number of EST replicates made it possible to derive curated DNA sequence with higher confidence and accuracy than from the sequencing of individual clones. The family has a core motif of 35 invariant and conserved amino acids centered on a single transmembrane span. Features of each predicted protein product were compared, and tissue distributions were determined. The gene family was named FXYD (pronounced fix-id) in recognition of invariant amino acids in its signature motif. The abundant proteins are involved in the control of ion transport.

Amino Acid Sequence↗
PubMed2003

Proteome analysis of hairy root from Panax ginseng C.A. Meyer using peptide fingerprinting, internal sequencing and expressed sequence tag data.

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.

Databases, Protein↗
PubMed1993

Compact protein sequencer for the C-terminal sequence analysis of peptides and proteins.

We describe the construction of a compact protein sequencer designed specifically for the C-terminal sequence analysis of peptides and proteins. This sequencer has a vertical flow path and is equipped with a continuous flow reactor (CFR). The flow paths for the various reagents and solvents have been minimized. A unique feature of this instrument is the design of a quadrate valve (quad valve) which permits the delivery of four solvents or reagents to the conversion flask (CF). Combination of two of these quad valves in series permits the delivery of eight solvents and reagents to the CFR. The CF contains three inputs from the top, one for transfer of the contents of the CFR, one which is used as a vent, and one for input of solvents or reagents from the CF quad valve. The CF drains from the bottom, connecting to a switching valve which allows delivery either to a waste bottle or to an on-line HPLC. Another unique feature of this instrument is the design of an optical flow detector which permits injection of approximately 90% of the contents of the CF for HPLC analysis. The overall size of the instrument (11 w x 16.5 h x 23.5 d in.) is smaller than commercially available instruments for protein sequencing and represents the first time an instrument has been constructed specifically for C-terminal sequence analysis. The utility of this instrument is demonstrated with the C-terminal sequence analysis of protein samples noncovalently applied to Zitex strips and with a peptide covalently attached to carboxylic acid-modified polyethylene film.

Amino Acids↗
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↗
PubMed1997

Transfection of the inner cell mass and lack of a unique DNA sequence affecting the uptake of exogenous DNA by sperm as shown by dideoxy sequencing analogues.

PURPOSE: The purpose of this study was to determine whether exogenous DNA internalized into blastocysts after transference from DNA-carrier sperm are localized at the inner cell mass or trophoblast cells and to identify differences in uptake of exogenous DNA fragments by sperm due to unique DNA sequences. METHODS: Mouse blastocysts at the hatching stage were exposed to migrating human sperm cells carrying exogenous DNA fragments synthesized from the E6-E7 conserved gene regions of human papillomavirus (HPV) types 16 and 18. After an interaction period of 2 hr, the transfected blastocysts were washed several times to remove extraneous sperm and the blastocysts were dissected into groups of cells derived from the inner cell mass and trophoblasts. The cells were analyzed by polymerase chain reaction (PCR) for the presence of HPV DNA fragments. In the second part of the experiment, thawed donor (N = 10) sperm cells were pooled, washed, and divided into two fractions. The first (control) fraction was added with formalin and further divided and added with a 35S-radiolabeled G, A, T, or C sequencing mixture. The second fraction was similarly treated but the formalin step was omitted from the treatment. After an hour of incubation at 37 degrees C, the sperm specimens were washed several times by centrifugation and DNA extracted by the GeneReleaser method. The extracted DNA were processed on sequence gels, and the autoradiographs analyzed. RESULTS: Mouse blastocysts transfected by carrier sperm with DNA from HPV types 16 and 18 showed localization of the HPV DNA to both the inner cell mass and trophoblast cells. Negative controls consisting of untreated human sperm and untreated mouse blastocysts did not reveal any evidence of HPV DNA. The positive sperm control generated expected DNA fragments from HPV types 16 and 18. In the second experiment, the intensities of the DNA fragments in the G, A, T, and C columns from low to high molecular weights were not different from the positive control bands. Band intensities of the four sequencing columns were similar. Formalin pretreatment of the sperm inhibited uptake of the DNA fragments from the smallest to the largest DNA molecules. CONCLUSIONS: Exogenous DNA taken into blastocysts are localized to both the inner cell mass and trophoblast cells. Only live sperm exhibited the capacity to carry various sizes of exogenous DNA, suggesting the involvement of active cell membrane mechanism in the transference process. The results showed that DNA fragments terminating in any of the four nucleotides were equally taken up by the sperm cell. Fragments of DNA produced by the sequencing reaction failed to identify a unique DNA sequence that would facilitate or inhibit the sperm from taking up exogenous DNA.

Animals↗
PubMed1983

Rat pancreatic ribonuclease: agreement between the corrected amino acid sequence and the sequence derived from its messenger RNA.

A corrected amino acid sequence of rat pancreatic ribonuclease is presented which is in agreement with the messenger RNA sequence in [J. Biol. Chem. (1982) 257, 14582-14585]. The corrections do not change the general position of rat ribonuclease in trees which can be constructed for ribonuclease sequences, although they do place the rat sequence somewhat closer to the mouse sequence. The evolutionary rate of ribonuclease in the rodent family of the Muridae (rat and mouse) now has been calculated to be 140 nucleotide substitutions per 10(8) years per 100 codons, and still is one of the highest rates yet observed. The occurrence of 4 additional amino acids at the C-terminus in several mammalian ribonucleases is in agreement with the position of a second stop codon in the 3' non-coding region of the rat messenger RNA sequence.

Amino Acid Sequence↗
PubMed1978

Nucleotide sequence of the DNA replication origin for human papovavirus BKV: sequence and structural homology with SV40.

DNA and RNA sequencing techniques were used to obtain the sequence surrounding the origin of DNA replication for human papovavirus BKV. The structure is characterized by a true palindrome of 17 residues followed by two sets of symmetrical sequences and a stretch of 20 AT residues. Within the two symmetrical sequences is a segment containing a strong purine bias, 23 of 26 nucleotides. These structures are similar, if not identical, to those found in the region of the SV40 replication, origin. Within the homologous DNA segments, 60-80% of the BKV and SV40 nucleotides are the same. The remarkable similarity of BKV and SV40 sequences containing the origins of DNA replication would appear to confirm our previous suggestion of an evolutionary relationship between the two genomes. In addition, topological similarities between these sequences suggest the possibility of certain structural requirements for bidirectional replication origins in these superhelical DNAs.

BK Virus↗
PubMed1985

A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA.

A simple new procedure was described for producing a sequential series of overlapping clones for use in DNA sequencing. The technique used single-stranded M13 DNA and complementary DNA oligomers to form specific cleavage and ligation substrates. It was, therefore, independent of the sequence of the DNA cloned into the vector. Deletions of varying sizes were generated from one end of the insert through the 3' to 5' exonuclease activity of T4 DNA polymerase. The approximate size of the deletion and therefore the starting point for DNA sequencing could be estimated by electrophoresis of the subcloned phage DNA on a agarose gel. This greatly reduced the number of templates that must be sequenced to obtain a complete sequence. The entire procedure could be carried out in one tube in less than a day. The procedure was used to subclone and sequence the maize mitochondrial 18 S rDNA and 5' flanking region (2622 bases) in less than a week. Other applications of oligomers and single-stranded DNA in the construction of insertions, deletions, and cDNAs are discussed.

Base Sequence↗
PubMed1983

Sequence organisation in nuclear DNA from Physarum polycephalum. Arrangement of highly-repeated sequences.

Recombinant plasmids containing highly repetitive Physarum DNA segments were identified by colony hybridisation using a radioactively-labelled total Physarum DNA probe. A large number of these clones also hybridised to a foldback DNA probe purified from Physarum nuclear DNA. The foldback DNA probe was characterised by reassociation kinetic analysis. About one-half of this component was shown to consist of highly repeated sequences with a kinetic complexity of 1100 bp and an average repetition frequency of 5200. Direct screening of 67 recombinant plasmids for foldback sequences using the electron microscope revealed that about one-half were located in segments of DNA containing highly repetitive sequences; the remainder were present in clones containing low-copy number repeated elements. Analysis of two DNA clones showed that they contained repetitive elements located in over half of all DNA segments containing highly repetitive DNA and that the foci containing these highly repetitive sequences had different sequence arrangements. The results are consistent with the hypothesis that the most highly repeated DNA sequence families in the Physarum genome are few in number and are clustered together in different arrangements in about one-sixth of the genome. Over one-half of the foldback DNA complement in the Physarum genome is derived from these segments of DNA.

DNA, Fungal↗
PubMed1988

Transposition of the Streptococcus lactis ssp. lactis Z270 lactose plasmid to pVA797: demonstration of an insertion sequence and its relationship to an inverted repeat sequence isolated by self-annealing.

The lactose plasmid pUCL22 of the single plasmid strain Streptococcus lactis ssp. lactis Z270 was demonstrated to fuse with the heterologous conjugative plasmid pVA797. The fusion of pUCL22 with pVA797 occurred by recombination between a specific sequence of pUCL22 and different sites of pVA797. The cointegrates of pUCL22::pVA797 were unstable: in the absence of lactose selection, they segregated plasmids that corresponded to pVA797 enlarged by one sequence of 1.2 kb, common to all derivative plasmids. This resolution sequence (RS) was shown to originate in the 9.7 kb BstEII restriction fragment of pUCL22 and to duplicate during replicon fusion. In addition, after nuclease S1 treatment of pUCL22 DNA, a self-annealing sequence was isolated; the two copies of this inverted repeat (IR) sequence were located on the 18 kb BamHI segment of the plasmid. This latter sequence was distinct from the RS with which it hybridized weakly. The RS was responsible for the transposition of the entire lactose plasmid; the role of the IR remains to be elucidated.

Conjugation, Genetic↗
PubMed1981

Nucleotide-sequence heterogeneity and sequence rearrangements in influenza virus cDNA.

Double-stranded cDNA has been synthesized from influenza virus RNA and cloned into derivatives of the bacteriophage M13 for sequence analysis. The characterization of over 200 clones has permitted an analysis both of nucleotide sequence heterogeneity and of clones containing unusual rearrangements of sequence. Heterogeneity, due to genetic variability in the RNA population and to in vitro synthetic errors, was detected at the low level of one nucleotide difference per 3 700 nucleotides. By contrast, gross sequence rearrangements were identified in eight clones. Inversions of sequence within the same cDNA molecule were the predominant type of rearrangement, and three mechanisms for producing such inversions are discussed. In addition, we observed rarer clones containing sequence from one RNA molecule joined to that from another molecule.

Base Sequence↗
PubMed1986

Covalently linked sequencing primer linkers (splinkers) for sequence analysis of restriction fragments.

A new method for direct sequence analysis of DNA restriction fragments uses synthetic covalently linked complementary oligodeoxynucleotides, as universal sequencing primer linkers (splinkers). These splinkers were designed to contain an inverted repeat sequence which forms a double-stranded hairpin structure with a known restriction site. The splinkers were characterized by their ability to be self-ligating (dimerized) and by their restriction digest product analysis of both the monomer and dimer. They can also be ligated to dephosphorylated DNA restriction fragments which contain the appropriate end. This was evidenced by mobility shifts of the splinker-ligated restriction fragments. The splinker-ligated restriction fragments, after denaturation, form a single-stranded DNA template containing an inverted repeat sequence (from splinker) at one terminus. The splinker is thus suitably oriented and serves as a primer for dideoxy nucleotide (nt) sequencing catalyzed by either Klenow fragment of Escherichia coli DNA polymerase I or avian myeloblastosis virus reverse transcriptase. As demonstrated for both pBR322 and phi X174, release of the primer extension strand by digestion at the splinker restriction site results in a ladder of labelled fragments which corresponds to a unique nt sequence.

Base Composition↗
PubMed1980

Separation and sequencing of the sequence isomers of pyrimidine deoxypentanucleoside tetraphosphates by high-performance liquid chromatography.

Chemical synthesis of DNA fragments with a defined sequence is extremely long and complex. A simpler alternative is isolation of fragments from partially hydrolysed DNA. Mixtures of sequence isomers of pyrimidine oligonucleotides were recently isolated on a preparative scale from hydrolysates of depurinated herring sperm DNA. These, however, could not be further separated by conventional techniques. Using high-performance liquid chromatography (HPLC), sequence isomers of pyrimidine di-, tri- and tetranucleotides were successfully separated and sequenced. In the present work, separation and sequencing of sequence isomers of pyrimidine pentanucleotides by HPLC was achieved, demonstrating the applicability of this particular approach in preparation of desired DNA fragments.

Animals↗
PubMed2003

Expressed sequence tags: alternative or complement to whole genome sequences?

Over three million sequences from approximately 200 plant species have been deposited in the publicly available plant expressed sequence tag (EST) sequence databases. Many of the ESTs have been sequenced as an alternative to complete genome sequencing or as a substrate for cDNA array-based expression analyses. This creates a formidable resource from both biodiversity and gene-discovery standpoints. Bioinformatics-based sequence analysis tools have extended the scope of EST analysis into the fields of proteomics, marker development and genome annotation. Although EST collections are certainly no substitute for a whole genome scaffold, this "poor man's genome" resource forms the core foundations for various genome-scale experiments within the as yet unsequenceable plant genomes.

Computational Biology↗
PubMed1980

Use of oligodeoxynucleotide primers to determine poly(adenylic acid) adjacent sequences in messenger ribonucleic acid. 3'-Terminal noncoding sequence of bovine growth hormone messenger ribonucleic acid.

Twelve synthetic oligodeoxynucleotide primers of the general sequence d(pT8-N-N') were tested in a reverse transcriptase reaction for specific initiation of complementary deoxyribonucleic acid (cDNA) synthesis at the poly(adenylic acid) junction of a messenger ribonucleic acid (mRNA) template. Only the sequence d(pT8-G-C) functioned as a specific primer of cDNA synthesis with an enriched fraction of bovine growth hormone mRNA from the anterior pituitary gland and produced unique fragments in a dideoxy sequencing reaction. The nucleotide sequence obtained by this method extended into the protein coding region of bovine growth hormone mRNA and was confirmed by chemical sequencing of the cDNA initiated with [5'-32P]d(pT8-G-C). The 3'-untranslated region of bovine growth hormone mRNA is 104 nucleotides in length and contains regions of significant homology with both rat and human growth hormone mRNAs, including the region surrounding the common AAUAAA hexanucleotide. The method presented here for selection of the d(pT8-N-N') primer complementary to the poly(A) junction of mRNA is of general applicability for nucleotide sequence analysis of partially purified mRNAs.

Animals↗
PubMed1999

High-resolution sequence typing of HLA-DQA1 and -DQB1 exon 2 DNA with taxonomy-based sequence analysis (TBSA) allele assignment.

High-resolution DNA sequencing of exon 2 of DQA1 and DQB1 genes that uses a taxonomy-based sequence analysis (TBSA) method to assign alleles was developed. The system uses fewer primers for polymerase chain reaction (PCR) amplification and sequencing than other methods and yields accurate DQA1 and DQB1 typing when either homozygous or heterozygous DNA samples are tested. The approach was initially corroborated by the correct typing of 10 blinded samples that had been previously typed by PCR using sequence-specific oligonucleotide probes (PCR-SSOP) or serology, and subsequently confirmed by sequencing of cloned PCR products. DNA from peripheral blood cell samples of 130 individuals enrolled in a case-control analysis of HLA determinants of abdominal aortic aneurysm were subsequently evaluated. Overall, 8 different DQA1 and 19 DQB1 alleles were identified. All 21 DQA1 heterozygous combinations and 45 of 49 DQB1 heterozygous combinations were successfully resolved with TBSA. The two pairs of heterozygous DQB1 combinations that were not unambiguously typed required sequence specific PCR amplification for correct allele identification. We conclude that the method provides precise analysis for HLA-DQ typing.

DNA↗
PubMed1978

Amino acid sequences of alpha-helical segments from S-carbosymethylkerateine-A. Complete sequence of a type-I segment.

The amino acid sequence of a type-I helical segment from the low-sulphur protein (S-carboxymethylkerateine-A) of wool was determined by combining automatic and manual-sequencing data. Whereas in the type-II helical segment most of the cationic groups occur in pairs, 11 of the 22 anionic residues in the sequence of the type-I segment were situated next to a second anionic residue. This suggests possible interactions between type-I and type-II helical segments in alpha-keratin. As observed with the sequence of a type-II helical segment a model constructed on 3.6 residues per turn of helix shows a line of hydrophobic residues along the helix, thereby supporting the physicochemical evidence that the molecule is predominantly helical and forms part of a coiled-coil structure. Examination of the sequence data by predictive methods indicates the possibilty of extensive sections of alpha-helix interspersed with discontinuities. The molecule contains a number of regions with peptide sequences identical with those found by other workers after enzymic digestion of fractions from oxidized wool.

Amides↗
PubMed1979

The isolation and partial sequence of peptides produced by cyanogen bromide cleavage of calf thymus non-histone chromosomal high-mobility-group protein 2. Sequence homology with non-histone chromosomal high-mobility-group protein 1.

Peptides produced by CNBr cleavage of non-histone chromosomal protein HMG 2 (CNBr peptides) were isolated and characterized, and their partial sequences were determined. The present sequence data account for over half of the sequence of the protein HMG (high-mobility-group) 2 molecule, and, together with previously published results, provide interesting information on the charge distribution within the molecule. Comparison of the CNBr-peptide-sequence data for protein HMG 2 with the previously published data on the CNBr peptides from protein HMG 1 reveals extensive sequence homology between the two proteins. Detailed evidence for the amino acid-sequence data has been deposited as Supplementary Publication SUP 50095 (6 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1978) 169, 5.

Amino Acid Sequence↗