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PubMed2005

New complete mitochondrial DNA sequence of the lancelet Branchiostoma lanceolatum (Cephalochordata) and the identity of this species' sequences.

Three mitochondrial (mt) genes were sequenced for two Atlantic lancelet species, Branchiostoma lanceolatum and B. floridae, to examine a serious discrepancy among previously published results of molecular studies: substantial sequence difference in a nuclear gene vs. virtual identity in the mt genome sequence. The results revealed that three mt genes of B. lanceolatum, collected from Helgoland in the North Sea and Naples in the Mediterranean, were quite diverged from those of B. floridae, collected from Tampa Bay, Florida. Therefore, the previously recognized identity in the mt genome between the two species is attributable to misidentification of materials used. To correct this misleading information, the complete mtDNA sequence of B. lanceolatum was determined for an individual from Helgoland.

Animals↗
PubMed1993

When acoustic sequences are not perceptual sequences: the global perception of auditory patterns.

Warren, Bashford, and Gardner (1990) found that when sequences consisting of 10 40-msec steady-state vowels were presented in recycled format, minimal changes in order (interchanging the position of two adjacent phonemes) produced easily recognizable differences in verbal organization, even though the vowel durations were well below the threshold for identification of order. The present study was designed to determine if this ability to discriminate between different arrangements of components is limited to speech sounds subject to verbal organization, or if it reflects a more general auditory ability. In the first experiment, 10 40-msec sinusoidal tones were substituted for the vowels; it was found that the easy discrimination of minimal changes in order is not limited to speech sounds. A second experiment substituted 10 40-msec frozen noise segments for the vowels. The succession of noise segments formed a 400-msec frozen noise pattern that cannot be considered as a sequence of individual sounds, as can the succession of vowels or tones. Nevertheless, listeners again could discriminate between patterns differing only in the order of two adjacent 40-msec segments. These results, together with other evidence, indicate that it is not necessary for acoustic sequences of brief items (such as phonemes and tones) to be processed as perceptual sequences (that is, as a succession of discrete identifiable sounds) for different arrangements to be discriminated. Instead, component acoustic elements form distinctive "temporal compounds," which permit listeners to distinguish between different arrangements of portions of an acoustic pattern without the need for segmentation into an ordered series of component items.(ABSTRACT TRUNCATED AT 250 WORDS)

Attention↗
PubMed1999

[De novo cancer and adenoma-carcinoma sequence of the colorectum--clinicopathological differences between de novo carcinoma and carcinoma with the sequence].

The adenoma-carcinoma sequence ("The great majority of colorectal carcinomas arises from adenomas") proposed by Morson et al (1972) has been generally accepted world wide. Considering the sequence from the viewpoint of human carcinogenesis in general, however, a few contradictions and mysterious phenomena are found in the development and growth process of the colorectal carcinomas. It is evident that the histogenesis of the adenoma-carcinoma sequence including such contradictions is logically false. Consequently, we should fundamentally reconsider the histogenesis of colorectal carcinoma. The premise for the induction of the histogenesis of colorectal carcinoma is that carcinomas are much more objectively differentiated from adenomas with severe atypia, and that hyperplastic glands with slight atypia are differentiated from adenomas with slight atypia. In order to objectify the histological interpretation of atypical grades, conversion of complicated histological designs into real numbers is required. Two discriminant functions with two variables (N/C ratio and density of tubuli in a unit area) for differentiating carcinoma from adenoma have been determined using computed image processing. Based on the discriminant functions for differentiating objectively between carcinoma, adenoma, and hyperplasia, the histogenesis of colorectal carcinomas has been deduced as follows: 70-80% of colorectal carcinomas arise from the colorectal mucosa (de novo carcinoma) and 20-30% arise from adenoma. Observing the various clinicopathological phenomena in colorectal adenomas and carcinomas from the viewpoint of histogenesis, the contradictions do not enter the logically constructed model. Furthermore, the biological behavior of de novo carcinoma is clinicopathologically different from that of carcinoma with the sequence.

Adenoma↗
PubMed1998

[Neuronal activity of monkey dorso-lateral premotor cortex during tasks of figure recognition guided motor sequence vs memorized spatial motor sequence].

In the last 2-3 decades the role of the premotor cortex (PM) of monkey in memorized spatial sequential (MSS) movements has been amply investigated. However, it is as yet not known whether PM participates in the movement sequence behaviour guided by recognition of visual figures (i.e. the figure-recognition sequence, FRS). In the present work three monkeys were trained to perform both FRS and MSS tasks. Postmortem examination showed that 202 cells were in the dorso-lateral premotor cortex. Among 111 cells recorded during the two tasks, more than 50% changed their activity during the cue periods in either task. During the response period, the ratios of cells with changes of firing rate in both FRS and MSS were high and roughly equal to each other, while during the image period, the proportion in the FRS (83.7%) was significantly higher than that in the MSS (66.7%). Comparison of neuronal activities during same motor sequence of two different tasks showed that during the image periods PM neuronal activities were more closely related to the FRS task, while during the cue periods no difference could be found. Analysis of cell responses showed that the neurons with longer latency were much more in MSS than in FRS in either cue or image period. The present results indicate that the premotor cortex participates in FRS motor sequence as well as in MSS and suggest that the dorso-lateral PM represents another subarea in function shared by both FRS and MSS tasks. However, in view of the differences of PM neuronal responses in cue or image periods of FRS and MSS tasks, it seems likely that neural networks involved in FRS and MSS tasks are different.

Animals↗
PubMed1999

Human phosphoribosylformylglycineamide amidotransferase (FGARAT): regional mapping, complete coding sequence, isolation of a functional genomic clone, and DNA sequence analysis.

Purines play essential roles in many cellular functions, including DNA replication, transcription, intra- and extra-cellular signaling, energy metabolism, and as coenzymes for many biochemical reactions. The de-novo synthesis of purines requires 10 enzymatic steps for the production of inosine monophosphate (IMP). Defects in purine metabolism are associated with human diseases. Further, many anticancer agents function as inhibitors of the de-novo biosynthetic pathway. Genes or cDNAs for most of the enzymes comprising this pathway have been isolated from humans or other mammals. One notable exception is the phosphoribosylformylglycineamide amidotransferase (FGARAT) gene, which encodes the fourth step of this pathway. This gene has been cloned from numerous microorganisms and from Drosophila melanogaster and C. elegans. We report here the identification of a human cDNA containing the coding region of the FGARAT mRNA and the isolation of a P1 clone that contains an intact human FGARAT gene. The P1 clone corrects the purine auxotrophy and protein deficiency of Chinese hamster ovary (CHO) cell mutants (AdeB) deficient in both the activity and the protein for FGARAT. The P1 clone was used to regionally map the FGARAT gene to chromosome region 17p13, a location consistent with our prior assignment of this gene to chromosome 17. A comparison of the DNA sequence of the human FGARAT and FGARAT DNA sequence from 17 other organisms is reported. The isolation of this gene means that DNA clones for all the 10 steps of IMP synthesis have been isolated from humans or other mammals.

Amino Acid Sequence↗
PubMed1996

Hypermethylation of calcitonin gene regulatory sequences in human breast cancer as revealed by genomic sequencing.

DNA methylation has been studied intensively during the past years in order to elucidate its role in the regulation of gene expression, gene imprinting and cancer progression. Earlier studies have shown that a general genomic under-methylation is associated with chronic lymphocytic leukemia and metastatic prostate cancer. Site-specific methylation changes, as revealed by the use of methylation-sensitive restriction enzymes, have been reported to occur in the promotor region of the calcitonin gene in chronic myeloid leukemia as it progresses from the chronic phase to blast crisis, in non-Hodgkin's lymphoid neoplasms and in non-lymphocytic leukemia. We have now explored possible methylation changes associated with benign and malignant breast tumors. Two approaches were employed: (i) chemical determination of general genomic methylation status and (ii) base-specific analysis of the methylation changes in the promoter of the calcitonin gene with the aid of genomic sequencing. The results did not reveal any changes of total DNA 5-methylcytosine content in ductal carcinoma of breast in comparison with benign tumors. There was a small, yet significant, increase in 5-methylcytosine content in lobular carcinoma. Genomic sequencing of the promoter region of the calcitonin gene, however, revealed a striking hypermethylation at or around the transcription start site of the gene in ductal carcinomas. In benign tumors and lobular carcinomas, this region was either entirely unmethylated or only slightly methylated. The latter changes may reflect a regional hypermethylation of the short arm of chromosome 11, which harbors, in addition to the calcitonin gene, a number of putative or established tumor-suppressor genes. Our results demonstrate that genomic sequencing in its present form can be used for a reliable and precise DNA methylation analysis of primary human tumors.

5-Methylcytosine↗
PubMed1992

Preferential usage of JH2 in D-J joinings with DQ52 is determined by the primary DNA sequence and is largely dependent on recombination signal sequences.

Rearrangement of DQ52 with JH in IgH genes takes place in a large part of lymphocytes at a very early stage of differentiation although usage of DQ52 seems to be limited in the functional IgH genes of mature B cells. A possible and likely explanation for this, is secondary D-JH joinings occurring after the initial DQ52-JH joinings, which becomes possible only when DQ52 segments join 5'-located JH segments. Previously we reported the non-random use of JH in joinings with DQ52, in which DQ52-JH2 joinings are dominant. We questioned how the preferential usage of JH2 in DQ52-JH joinings is determined by utilizing extrachromosomal DNA substrates. Extrachromosomal DNA substrates with a combination of DQ52 and multiple JH segments were prepared. These plasmids with a polyoma-derived replication system were transfected into pre-B cell lines and recombination profiles of recovered plasmids were analyzed semi-quantitatively. Experiments with plasmids containing all four JH segments in normal configuration showed an apparent high frequency of JH2 usage, similar to the rearrangement previously observed in thymocyte genomes. In plasmids containing two JH segments, when one was JH2, it was rearranged more frequently than the other, independent of its proximity to DQ52. The JH3 with recombination signal sequences (RSS) substituted for JH2 was rearranged more frequently than the naive JH2 itself indicating the importance of RSS. A JH3 hybrid with the 200 bp upstream sequence of JH2 but excluding RSS, however, failed to increase JH3 use. The addition of one nucleotide to the 22-bp spacer of JH1 RSS increased the frequency of JH1 usage. These observations suggest that the preferential usage of JH2 is determined by the primary DNA sequence and is largely dependent on RSS.

Animals↗
PubMed1996

The limiting mobility of DNA sequencing fragments for both cross-linked and noncross-linked polymers in capillary electrophoresis: DNA sequencing at 1200 V cm-1.

The mobility of DNA sequencing fragments was measured in Long-Ranger gels at an electric field ranging from 200 to 1200 V cm-1 and in noncross-linked polyacrylamide at electric fields ranging from 100 to 300 V cm-1. In both cases, N*, the fragment length that denotes the onset of biased reptation with orientation, is inversely proportional to electric field. The inverse dependence of N* is inconsistent with the original biased reptation model but is consistent with modern models of DNA migration. While separation speed increases dramatically with electric field, the number of bases determined in a separation decreases in proportion to field strength. We present a DNA sequencing run at an electric field of 1200 V cm-1. Roughly 200 bases of sequence are determined in 3.5 min.

Base Sequence↗
PubMed1995

Analysis of the joining sequences of the t(15;17) translocation in human acute promyelocytic leukemia: sequence non-specific recombination between the PML and RARA genes within identical short stretches.

Molecular analysis of the t(15;17) translocation in 70 patients with acute promyelocytic leukemia (APL) confirmed that the breakpoints of chromosome 15 were located in two regions of the promyelocytic leukemia (PML) gene, mainly introns 3 and 6, whereas the breakpoints of chromosome 17 were consistently in intron 2 of the retinoic acid receptor alpha (RARA) gene. To study the reason for the clustering of the breakpoints and the underlying mechanism of the chromosomal translocation, we characterized the joining sequences of der(15) and der (17) by polymerase chain reaction in samples from eight patients with APL. There was no cluster of the breakpoints within the introns, and no consensus sequence-motif was found around them. One or nine extra nucleotides were inserted into two joining sites. There were identical stretches of one to seven nucleotides between the PML and RARA genes in the majority of the joining sequences. These data provide a potential model of the t(15;17) translocation: random DNA double strand cleavage, modification of DNA ends by enzymes including terminal deoxynucleotidyl transferase, and single strand base-pairing within identical short stretches. Furthermore, APL develops only when the PML and RARA genes are rearranged, within restricted genomic regions and a functional PML-RARA chimeric product is produced, and this might lead to a clustering of the breakpoints.

Base Sequence↗
PubMed1984

Amino acid sequence of the carboxy-terminal part of an acidic type I cytokeratin of molecular weight 51 000 from Xenopus laevis epidermis as predicted from the cDNA sequence.

The DNA sequence of a clone from a cDNA library made from Xenopus laevis skin is described. This sequence represents the 3'-terminal end of an mRNA which codes for an epidermal cytokeratin polypeptide of mol. wt. 51 000 of the acidic (type I) subfamily as identified by hybridization-selection of mRNAs, followed by gel electrophoretic identification of the polypeptides synthesized by translation in vitro. The partial amino acid sequence of the amphibian cytokeratin shows strong similarity to type I cytoskeletal keratins from human (mol. wt. 50 000) and murine (mol. wt. 59 000) epidermis. In the non alpha-helical tail region the human and the non-mammalian (Xenopus) keratins are more similar to each other than to the murine protein, indicating that the former are equivalent cytokeratin polypeptides and belonging to a special subclass of type I keratin polypeptides devoid of glycine-rich regions in the carboxy-terminal portion. The evolutionary conservativity of the genes coding for cytokeratins is discussed.

Amino Acid Sequence↗
PubMed1986

Molecular cloning and sequencing of the human erythrocyte 2,3-bisphosphoglycerate mutase cDNA: revised amino acid sequence.

The human erythrocyte 2,3-bisphosphoglycerate mutase (BPGM) is a multifunctional enzyme which controls the metabolism of 2,3-diphosphoglycerate, the main allosteric effector of haemoglobin. Several cDNA banks were constructed from reticulocyte mRNA, either by conventional cloning methods in pBR322 and screening with specific mixed oligonucleotide probes, or in the expression vector lambda gt 11. The largest cDNA isolated contained 1673 bases [plus the poly(A) tail], which is slightly smaller than the size of the intact mRNA as estimated by Northern blot analysis (approximately 1800 bases). This cDNA encodes for a protein of 258 residues; the protein yielded 34 tryptic peptides which were subsequently isolated by h.p.l.c. Our nucleotide sequence data were entirely confirmed by the amino acid composition of these tryptic peptides and reveal several major differences from the published sequence; the revised amino acid sequence of human BPGM is presented. These findings represent the first step in the study of the expression and regulation of this enzyme as a specific marker of the erythroid cell line.

Amino Acid Sequence↗
PubMed2005

Assigning new GO annotations to protein data bank sequences by combining structure and sequence homology.

Accompanying the discovery of an increasing number of proteins, there is the need to provide functional annotation that is both highly accurate and consistent. The Gene Ontology (GO) provides consistent annotation in a computer readable and usable form; hence, GO annotation (GOA) has been assigned to a large number of protein sequences based on direct experimental evidence and through inference determined by sequence homology. Here we show that this annotation can be extended and corrected for cases where protein structures are available. Specifically, using the Combinatorial Extension (CE) algorithm for structure comparison, we extend the protein annotation currently provided by GOA at the European Bioinformatics Institute (EBI) to further describe the contents of the Protein Data Bank (PDB). Specific cases of biologically interesting annotations derived by this method are given. Given that the relationship between sequence, structure, and function is complicated, we explore the impact of this relationship on assigning GOA. The effect of superfolds (folds with many functions) is considered and, by comparison to the Structural Classification of Proteins (SCOP), the individual effects of family, superfamily, and fold.

Algorithms↗
PubMed1994

Quantitative DNA sequencing to determine the relative protein-DNA binding constants to multiple DNA sequences.

DNA sequencing technology was modified into a quantitative assay, which for multiple DNA sequences allowed the simultaneous determination of relative protein-DNA binding constants. The band mobility shift of the protein-DNA binding reactions partitions the mixture of DNA sequences into bound and unbound fractions. The quantitation of that partitioning gives directly the relative binding constants, usually with accuracies of better than +/- 20%. The protein of interest was the Mnt repressor of Salmonella bacteriophage P22, and the synthetic DNA contained Mnt's natural operator with a randomized position.

Adenosine Triphosphate↗
PubMed1995

Selection of high-affinity binding sites for sequence-specific, DNA binding proteins from random sequence oligonucleotides.

We describe a rapid and sensitive method to isolate sets of high-affinity binding sites for sequence-specific DNA binding proteins. The DNA binding domain of the protein is expressed in Escherichia coli as a fusion with glutathione S-transferase (GST). Binding reactions are set up with total soluble extract from induced bacteria and a double-stranded oligonucleotide for which the central 32 bp have been randomized. To ensure stringent conditions, binding is done in the presence of high levels of poly(dI:C). The GST fusion protein is recovered by the addition of glutathione-Sepharose. Following extensive washing of the Sepharose beads, the bound oligonucleotides are rescued by polymerase chain reaction amplification. The amplified material is used in the next cycle of selection and amplification. Approximately five cycles are needed to obtain a pure population of high-affinity sites, which are then cloned and sequenced. This procedure should be applicable to any sequence-specific DNA binding protein for which the cDNA is available and which can be expressed in bacteria in a functional form.

Base Sequence↗
PubMed1999

HIF1A gene transcription is dependent on a core promoter sequence encompassing activating and inhibiting sequences located upstream from the transcription initiation site and cis elements located within the 5'UTR.

Hypoxia inducible factor-1 (HIF-1) is a transcription factor composed of two subunits, HIF-1alpha and ARNT, which is activated under hypoxia. HIF-1alpha mRNA is expressed constitutively in a wide variety of cell types, whereas in some others HIF1A gene expression is upregulated by hypoxia. In this report, we show that in endothelial cells (HMEC-1) the HIF-1alpha mRNA expression level is the same in both normoxia and hypoxia. Deletion analysis experiments of the HIF1A promoter showed that in hypoxia HIF1A gene expression is upregulated through a short sequence located next to the transcription initiation site. We also show that in hypoxia another sequence located upstream from the +1 initiation site plays an inhibitory role on HIF1A transcription in HMEC-1 but not in hepatoma cells and brings back this expression level to that observed in normoxia. Finally, we demonstrate that HIF1A gene transcription is dependent on Sp1 binding sites and that the 5'UTR sequence also contains other important cis-acting elements.

5' Untranslated Regions↗
PubMed1994

DNA sequence GCGAATGAGC containing the human centromere core sequence GAAT forms a self-complementary duplex with sheared G.A pairs in solution.

The DNA sequence dGCGAATGAGC has a well-resolved, two-dimensional nuclear Overhauser (NOESY) spectrum that is suitable for high quality solution structure determination by NMR methods; in solution this sequence forms a stable self-complementary duplex containing sheared G.A base-pairs. A total of 220 distance constraints derived from time-dependent NOE measurements were collected and refined by repeated back-calculation of the NOESY spectra. Distance information from imino proton studies and from exclusive two-dimensional correlated spectroscopy (E. COSY) and/or linewidth analysis was included in the structure calculation using the program DSPACE 4.2, followed by restrained energy minimization with the program DISCOVER using the AMBER force field. The energies of the distance geometry (DG) structures decreased rapidly in the first few cycles and approached -510 +/- 3 kcal after 1000 cycles of conjugate gradient minimization (about 540 kcal lower than in the initial DG structures). All 15 final DG structures converged to a single family of closely related structures with pair-wise r.m.s.d. values of 0.96 +/- 0.34 A, which was further reduced by energy minimization to 0.70 +/- 0.35 A. Rather unusual structural features of the duplex are revealed in the final structures. The results indicate that, in addition to normal sequences with standard base-pairing, unusual nucleic acid structures can also be determined in solution with quite high precision by NMR/distance geometry methods.

Base Composition↗
PubMed1996

Nucleosome DNA sequence pattern revealed by multiple alignment of experimentally mapped sequences.

Five different algorithms have been applied for detecting DNA sequence pattern hidden in 204 DNA sequences collected from the literature which are experimentally found to be involved in nucleosome formation. Each algorithm was used to perform a multiple alignment of the nucleosome DNA sequences within the window 145 nt, the size of a nucleosome core DNA. From these alignments five pairs of AA and TT dinucleotide positional frequency distributions have been computed. The frequency profiles calculated by different algorithms are rather different due to substantial noise. They, however, share several important features. Both AA and TT dinucleotide positional frequencies display periodicity with the period of 10.3(+/- 0.2) bases. TT dinucleotides appear to be distributed symmetrically relative to AA dinucleotides of the same DNA strand, with the center of symmetry at the midpoint of the nucleosome core DNA. The phase shift between the AA and TT patterns is about 6 bp. Superposition of the five pairs of the AA (TT) positional frequency profiles has produced the refined pattern, with the above features well pronounced. An interesting novel feature of the pattern is an absence of central peaks in the periodical AA and TT distributions. This may indicate that the central section of nucleosome DNA, 15 bp around the dyad axis of the nucleosome, is not bent. Positional distributions of other dinucleotides were not found in this study to be as informative as the ones for AA and TT.

Algorithms↗
PubMed1986

Nucleotide sequence of the BamHI repetitive sequence, including the HindIII fundamental unit, as a possible mobile element from the Japanese monkey Macaca fuscata.

Clustered repeat units produced by BamHI digestion of genomic DNA from the Japanese monkey Macaca fuscata [JMr(BamHI)] were sequenced by dideoxy DNA sequencing. The nucleotide sequences of several individual repeats showed that the BamHI repeat contains the 170-bp HindIII element as an integral part, and that it has more than 90% homology with the HindIII repeat element [AGMr(HindIII)] found in the genomic DNA of the African green monkey. In the JMr(BamHI) repeat unit, the 170-bp HindIII element is flanked by a 6-bp inverted repeat, which is part of a 22-bp direct repeat. This latter repeat of 22-bp asymmetrically overlaps the border between the internal AGMr(HindIII)-like region and adjacent regions of the JMr(BamHI) repeat. A similar structural feature of the BamHI repeat unit has been found in the genomic DNA of the baboon, but not in that of the African green monkey. These results show clearly that the BamHI repeat of the modern Japanese monkey originated as a result of insertion of an AGMr(HindIII) element into a certain site(s) of the genomic DNA of an ancestor of the modern Japanese monkey before Macaca-Cercocebus divergence.

Animals↗