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PubMed1983

DNA sequence adjacent to flagellar genes and evolution of flagellar-phase variation.

A variety of factors, including phase variation, are involved in the regulation of flagellin gene expression in Salmonella sp. Flagellar-phase variation refers to the alternate expression of two different flagellin genes, H1 and H2. Site-specific inversion of a DNA segment adjacent to the H2 gene is responsible for switching expression. The segment includes the H2 promoter as well as the hin gene, which is required to mediate the inversion. Sequences in this region have homology with the corresponding sequences adjacent to the H1 flagellin gene in Salmonella sp. and the hag flagellin gene in Escherichia coli. The hin gene has also been shown to be homologous to the gin gene, which is found on bacteriophage Mu. To understand gene expression and the origin of these relationships, we have compared the DNA sequence adjacent to all three flagellin genes. The sequence data suggest a mechanism for the evolution of the hin-H2 locus.

Amino Acid Sequence↗
PubMed1987

Diet composition and the plasma levels of some peptides regulating pancreatic secretion in the pig.

The aim of the present work was to study the effect of a modification of diet composition upon the plasma levels of some peptides known to be involved in the hormonal regulation of exocrine pancreas secretion. Six growing Large-White pigs weighing 41 +/- 3.2 kg were fitted with a catheter in a carotid artery; four of these pigs were also fitted with permanent fistulae in the pancreatic duct and duodenum. All the pigs were adapted to a control diet (C) during an 8-day period before surgery. In the 8-day postoperative period and a first experimental period of 4 days, they were fed on the same control diet. Three pigs were then fed the experimental diets in the following sequence: fat-rich diet (F) for 7 days, control diet (C) for 7 days, starch-rich diet (S) for 7 days, whereas the other three pigs were fed the same diets over the same time lengths but in inverse sequence: diet S, diet C, diet F. The three diets were isoproteinic (16% protein) and isocaloric (3,850 cal/kg). The pancreatic secretion and the plasma levels of cholecystokinin (CCK), secretin, pancreatic polypeptide (PP) and somatostatin were analysed during the 4 days of the first experimental period and the last day of each of the other three experimental periods. Total proteins and lipase and amylase activities were determined in pancreatic juice samples collected over the 7 hours following the morning meal. Arterial blood was sampled at 9 h 00 (before meal consumption), 9 h 30, 10 h 00 and every hour until 16 h 00. The results confirm pancreatic adaptation to the diet, i.e. increase of lipase specific activity (x 1.8) when the pigs ingested 6 times more fat (diet S----diet F) per day, and an increase in amylase specific activity (x 2.3) when they ingested 3 times more starch (diet F----diet S) per day. Furthermore, changes in diet composition did not lead to any durable, significant change in plasma peptide levels. In conclusion, CCK, secretin, PP and somatostatin, known to regulate exocrine pancreas secretion, would not be involved in the mechanisms of pancreatic amylase and lipase adaptation to the amount of carbohydrate and fat ingested by pigs.

Animals↗
PubMed1982

Incompletely base-paired flip-flop terminal loops link the two DNA strands of the vaccinia virus genome into one uninterrupted polynucleotide chain.

The nature of the ends of the vaccinia virus genome was determined by nucleotide sequencing. Our finding of terminal hairpins indicated that the linear double-stranded DNA molecule consists of a single continuous polynucleotide chain. The 104 nucleotide apex of the hairpin contains predominantly A and T residues and is incompletely based-paired. These loops exist in two forms, which when inverted with respect to each other are complementary in sequence. Both forms of the 104 nucleotide loop are present in nearly equimolar amounts of each end of the genome. A set of 13 tandem 70 bp repeats begins 87 bp from the proximal segment of the terminal loop, followed by a unique sequence of 325 bp, and then by a second set of 18 tandem 70 bp repeats. The sequence of the 70 bp repeats reveals a 13 bp internal redundancy. Self-priming and de novo start replication models, which involve a site-specific nick in one DNA strand proximal to the 104 nucleotide loop, account for the observed sequence inversions and incomplete base-pairing. Similar mechanisms may be involved in replication of the ends of the eucaryotic chromosome.

Base Sequence↗
PubMed1978

Instability of plasmid DNA sequences: macro and micro evolution of the antibiotic resistance plasmid R6-5.

Detailed examination of the structure of cloned DNA fragments of the R6-5 antibiotic resistance plasmid has revealed a substantial degree of polynucleotide sequence heterogeneity and indicates that sequence rearrangements in plasmids and possible other replicons occur more frequently than has hitherto been appreciated. The sequences changes in cloned R6-5 fragments were shown in some instances to have occurred prior to cloning, i.e. existing in the original population of R6-5 molecules that was obtained from a single bacterial clone and by several different criteria judged to be homogeneous, and in others to have occurred either during the cloning procedure or during subsequent propagation of hybrid molecules. The molecular changes that are described involved insertion/deletion of the previously characterized IS2 insertion element, formation of a new inverted repeat structure probably by duplication of a preexisting R6-5 DNA sequence, sequence inversion, and loss and gain of restriction endonuclease cleavage sites.

Anti-Bacterial Agents↗
PubMed1996

Mutational analysis of the nucleotide binding site of the epidermal growth factor receptor and v-Src protein-tyrosine kinases.

Tyrosine kinases differ from serine/threonine kinases in sequences located at the active site where ATP and substrate bind. In the structure of cyclic AMP-dependent protein kinase, the catalytic loop contains the sequence Lys-Pro-Glu where the Lys residue contacts the gamma-phosphate of ATP and the Glu residue contacts a basic residue located in the peptide substrate. In tyrosine kinases, the analogous sequence is Ala-Ala-Arg in the receptor tyrosine kinase subfamily and Arg-Ala-Ala in the Src tyrosine kinase subfamily. To deduce the role of these residues in tyrosine kinase function, site-directed mutations were prepared in the epidermal growth factor receptor (EGFR) and in v-Src and effects on ATP binding and kinase activity were determined. Changing Arg to either Lys or Ala dramatically reduced activity of both tyrosine kinases and this correlated with loss of ATP binding. Changing the orientation of this sequence impaired activity of EGFR to a greater extent than that of v-Src but did not change substrate specificity of the two enzymes. These results support the hypothesis that Arg functions to coordinate the gamma-phosphate of ATP. Analysis of sequence inversions in the catalytic loop indicate that the active site of v-Src exhibits greater flexibility than that of EGFR.

Adenosine Triphosphate↗
PubMed1998

Atomic force microscopy of crystalline insulins: the influence of sequence variation on crystallization and interfacial structure.

The self-association of proteins is influenced by amino acid sequence, molecular conformation, and the presence of molecular additives. In the presence of phenolic additives, LysB28ProB29 insulin, in which the C-terminal prolyl and lysyl residues of wild-type human insulin have been inverted, can be crystallized into forms resembling those of wild-type insulins in which the protein exists as zinc-complexed hexamers organized into well-defined layers. We describe herein tapping-mode atomic force microscopy (TMAFM) studies of single crystals of rhombohedral (R3) LysB28ProB29 that reveal the influence of sequence variation on hexamer-hexamer association at the surface of actively growing crystals. Molecular scale lattice images of these crystals were acquired in situ under growth conditions, enabling simultaneous identification of the rhombohedral LysB28ProB29 crystal form, its orientation, and its dynamic growth characteristics. The ability to obtain crystallographic parameters on multiple crystal faces with TMAFM confirmed that bovine and porcine insulins grown under these conditions crystallized into the same space group as LysB28ProB29 (R3), enabling direct comparison of crystal growth behavior and the influence of sequence variation. Real-time TMAFM revealed hexamer vacancies on the (001) terraces of LysB28ProB29, and more rounded dislocation noses and larger terrace widths for actively growing screw dislocations compared to wild-type bovine and porcine insulin crystals under identical conditions. This behavior is consistent with weaker interhexamer attachment energies for LysB28ProB29 at active growth sites. Comparison of the single crystal x-ray structures of wild-type insulins and LysB28ProB29 suggests that differences in protein conformation at the hexamer-hexamer interface and accompanying changes in interhexamer bonding are responsible for this behavior. These studies demonstrate that subtle changes in molecular conformation due to a single sequence inversion in a region critical for insulin self-association can have a significant effect on the crystallization of proteins.

Amino Acid Sequence↗
PubMed1992

Investigations on the mechanism of the salt-induced peptide formation.

The applicability of the salt-induced peptide formation in aqueous solution--the simplest model so far for peptide synthesis under primitive earth conditions--is demonstrated for valine as another amino acid, and the formation of mixed peptides in systems containing glycine, alanine and valine is investigated. The dominant dipeptides formed are Gly-Gly, Gly-Ala and Gly-Val, at longer reaction times sequence inversion produces Ala-Gly and, considerably slower, Val-Gly. Ala-Ala is also produced and the relative amounts of the diastereomers prove the high conservation of optical purity of the original amino acids over a considerable time. The results lead to some further conclusions about the reaction mechanism and the possible dominance of peptide sequences in primordial dipeptides.

Alanine↗
PubMed1993

Preliminary evaluation: magnetic resonance of urography using a saturation inversion projection spin-echo sequence.

A saturation inversion projection (SIP) spin-echo technique is reported which allows a reliable direct visualization of the urogenital system as well as its functional performance in magnetic resonance imaging. We used an imaging sequence with a 90 degree saturation pulse and two 180 degree inversion pulses followed by a short spin-echo (SE) pulse sequence. The three time intervals in the 90 degree-180 degree-180 degree-SE pulse train were adjusted to suppress the signals of soft tissues and fat. After intravenous injection of the contrast agent Gd-DTPA, a shortening of the kidney T1 and the T1 of the urogenital system is obtained below the T1 values of fat and soft tissues, and these remaining ultra-short T1 tissues were imaged with the SIP sequence. Using a sequential measuring technique a quantitative evaluation of the glomerular filtration rate seems to be possible with a time resolution of 18 sec per image. In addition, magnetic resonance urography using the SIP sequence provided a good visualization of the urogenital system and may show several clinical utilities in further clinical studies.

Contrast Media↗
PubMed1999

Molecular structure of canine LINE-1 elements in canine transmissible venereal tumor.

We determined the 4251-bp sequence of open reading frame 2 (ORF2) of canine LINE-1 retroposon that encodes 1275 amino acids. The truncated LINE-1 inserts associated with transmissible venereal tumor (TVT) of dogs contained the 1378-bp LINE-1 insert (TVT-LINE) flanked by 10-bp direct repeats upstream to c-myc gene. The TVT-LINE elements were composed of 416 bp inverse sequences homologous to the complementary strand of the LINE-1, a 5-bp deletion and 962-bp sequences homologous to the 3' region of the LINE-1.

Animals↗
PubMed1998

Evolution of neuronal changes in the course of Alzheimer's disease.

Alzheimer's disease entails multiple neuronal systems and results from neuronal cytoskeletal degeneration of only a few types of nerve cells. Essential for neuropathological diagnosis is assessment of the presence of neurofibrillary tangles and neuropil threads. The destructive process begins in predisposed cortical induction sites, thereafter invading other portions of the cerebral cortex and specific sets of subcortical nuclei in a predictable sequence with little variation. The location of the tangle-bearing neurons and severity of the pathology allow the distinction of six stages in disease propagation (transentorhinal I-II: clinically silent cases; limbic III-IV: incipient Alzheimer's disease; neocortical V-VI: fully-developed Alzheimer's disease). The pattern of appearance of the neurofibrillary changes bears a striking resemblance to the inverse sequence of cortical myelination. The average myelin content is a negative image of the density of intraneuronal lipofuscin deposits. Pigment-laden neurons endowed with a long, thin, and sparsely myelinated axon are prone to develop AD-related changes. The emergence of the first neurofibrillary changes, at whatever age these occur, signals the onset of a degenerative process that persists until death. An extended period of time elapses between the beginning of histologically verifiable lesions and the appearance of initial clinical symptoms. Once initiated, however, cytoskeletal deterioration inexorably progresses, and neither remission nor recovery is observed.

Alzheimer Disease↗
PubMed1991

Structural comparison of the B-DNA dodecamers d(CGCGTTAACGCG) and d(CGCGAATTCGCG) with T2A2 and A2T2 tracts.

The x-ray structure of the deoxy oligonucleotide dodecamer d(CGCGTTAACGCG) recently determined in our laboratory shows that the helical parameters of the central TTAA segment are significantly different compared to the central AATT in d(CGCGAATTCGCG). The roll in the central TA step of the T2A2 dodecamer opens towards the minor groove while the AT step of the A2T2 dodecamer opens towards the major groove. Also, the roll angles at the steps 4 and 8 (GT and AC in T2A2) and (GA and TC in A2T2) are in opposite directions. The high cup and helical twist angles at the central base-pair of T2A2 decreases the base stacking interactions compared to A2T2. Tilt angles within the tetranucleotide segments TTAA and AATT have opposite signs. In spite of the local differences caused by the sequence inversion (TTAA----AATT), the two dodecamers exhibit similar overall bending. The top third is more bent than the bottom third relative to the central segment. This asymmetric bending in the two dodecamers is mainly due to crystal packing interactions.

Base Sequence↗
PubMed2003

Gemcitabine and cisplatin versus vinorelbine and cisplatin versus ifosfamide+gemcitabine followed by vinorelbine and cisplatin versus vinorelbine and cisplatin followed by ifosfamide and gemcitabine in stage IIIB-IV non small cell lung carcinoma: a prospective randomized phase III trial of the Gruppo Oncologico Italia Meridionale.

PURPOSE: we carried out a phase III randomized trial to compare vinorelbine-cisplatin regimen to gemcitabine-cisplatin regimen, and to a sequential administration of gemcitabine-ifosfamide followed by vinorelbine-cisplatin or the opposite sequence of vinorelbine-cisplatin followed by ifosfamide-gemcitabine according to the 'worst drug rule' hypothesis in patients with locally advanced unresectable stage IIIB or metastatic stage IV non-small cell lung cancer. The primary endpoint was survival parameters, while secondary endpoints included analysis of response rates and toxicity. PATIENTS AND METHODS: patients were randomized to receive: (a) gemcitabine 1000 mg/m(2) on days 1, 8 and 15 plus ifosfamide 1500 mg/m(2) on days 8-12 with mesna uroprotection (GI regimen) followed by vinorelbine 25 mg/m(2) on days 1 and 8 plus cisplatin 100 mg/m(2) on day 1 (GI --> VC regimen); (b) the opposite sequence (VC --> GI); (c) vinorelbine plus cisplatin as above described (VC regimen); or (d) gemcitabine 1400 mg/m(2) on days 1 and 8 plus cisplatin 100 mg/m(2) on day 8 (GC regimen). All regimens were given every 4 weeks. All patients were chemotherapy naive and had a ECOG PS 0-2. RESULTS: 400 patients were enrolled into the trial. Interim analysis after inclusion of 243 patients showed that ORR were 19% in the GI --> VC arm, 32% in the inverse sequence arm (CV --> GI), 42% in the VC arm, and 30% in the GC arm. The VC arm was statistically superior over the GI --> VC arm (p = 0.0074), but not over the other regimens. Median TTP was 3.1 months in the GI --> VC arm versus 5.0 months in the VC --> GI arm (p = 0.014). For these reasons the GI --> VC and VC --> GI arm were closed since the 'worst drug rule' hypothesis was rejected. Accrual in the VC and GC arms continued up to 140 and 138 patients respectively. Final ORR were 44% for the VC regimen (4 CR), and 34% for the GC regimen (1 CR). This difference was statistically significant (p = 0.032). OS was 9.0 and 8.2 months, respectively, with no statistically significant difference. The 1-year survival rate was 24 and 20%, respectively for VC and GC regimens. As expected the incidence of phlebitis was higher in the VC arm, while thrombocytopenia, flu-like syndrome and asthenia were more frequent in the GC arm. CONCLUSIONS: the results of this trial indicate that the combination of vinorelbine and cisplatin and that of gemcitabine and cisplatin are equivalent in terms of median TTP and OS, although the vinorelbine-cisplatin regimen is associated with a higher ORR. Both regimens may be considered as reference treatments for future studies. Moreover, our data reject the 'worst drug rule' hypothesis of sequential treatments in NSCCL at least with the combination used in this study.

Adenocarcinoma↗
PubMed2006

Improvement of the inverse-gated-decoupling sequence for a faster quantitative analysis of various samples by 13C NMR spectroscopy.

The inverse-gated-decoupling sequence enables quantitative (1)H decoupled (13)C spectra to be obtained. We modified this sequence so as to obtain the same result in less time for molecules containing carbons with various relaxation properties. For that, we determined the optimal (13)C longitudinal-magnetization initial value for a faster relaxation while (1)H decoupler is stopped. This value can be calculated precisely via the nuclear Overhauser effects, the longitudinal relaxation times, together with the determination of the relaxation rate constants of carbons while (1)H are out of equilibrium. A supplementary delay of (1)H decoupling and/or a series of selective pulses applied at the beginning of the recovery delay allow an acceleration of (13)C longitudinal relaxation. We applied this method to the molecule of vanillin. The simultaneous quantification of all carbons was carried out with a recovery delay divided by two compared to the usual sequence.

Benzaldehydes↗
PubMed2003

The membrane-interactive tail of cytochrome b(5) can function as a stop-transfer sequence in concert with a signal sequence to give inversion of protein topology in the endoplasmic reticulum.

Sequence analyses of the C-terminal membrane intercalative region of the rat cytochrome b(5) indicated that this domain has, in addition to a signal sequence, a combined element of the classic stop-transfer sequence typically found in a variety of transmembrane proteins. Such bitopic protein arrangements arise by tandem but topogenically displaced activities of cleavable/noncleavable signal and stop-transfer sequences. A fusion precursor comprising an N-terminally linked prokaryotic signal sequence and the full-length of mammalian cytochrome b(5), including its C-terminal membrane insertion sequence, was engineered to investigate the outcome of this combination of signals on the targeting and topology of the cytochrome b(5) in the endoplasmic reticulum membrane. Precytochrome b(5) was cotranslationally translocated across the endoplasmic reticulum membrane. The signal-processed cytochrome b(5) was integrally anchored in the membrane with the globular domain facing the lumen. Thus, the topology of the signal sequence-directed cytochrome b(5) in the microsomal vesicle was reversed with respect to that of the native form. Posttranslational incubation of the precytochrome b(5) with microsomes resulted in a "loose" incorporation of the unprocessed form onto the surface of the vesicle. Our findings suggest that the membrane-insertion sequence of cytochrome b(5) has a functional stop-transfer sequence. We discuss the implications of these findings with respect to selective targeting of cytochrome b(5) to the endoplasmic reticulum membrane in the view that signal and stop-transfer sequences are often interchangeable or combined for topogenic functions.

Amino Acid Sequence↗
PubMed1999

Thermolysin and mitochondrial processing peptidase: how far structure-functional convergence goes.

The structure-functional convergence between two Zn-dependent proteases, namely thermolysin and mitochondrial processing peptidase (MPP), is described. These two families of nonhomologous enzymes show not only functional convergence of several active site residues as in chymotrypsin and subtilisin, but also structural convergence of overall molecular architectures including the beta-sheet arrangement and packing of the surrounding alpha-helices. The major functionally important structural elements are present in both enzymes with different topological connections and often in reverse main-chain orientation, but display similar packing. The structural comparison helps to rationalize sequence "inversion" of the HEXXH thermolysin consensus present as HXXEH in MPP. The described structural convergence may be due to a limited number of alternatives to build a Zn-protease that utilizes hydrogen bonding between a substrate main chain and the enzyme beta-sheet for substrate binding.

Amino Acid Sequence↗
PubMed1997

Characterization of CR1 repeat random PCR markers for mapping the chicken genome.

Polymerase chain reaction (PCR) primers complementary to portions of the chicken repetitive element CR1 have been used previously to generate useful markers on the chicken genome linkage map. To understand better the genetic basis for this technique and to convert CR1-PCR loci to markers useful in physical genome mapping, five polymorphic CR1-PCR-generated DNAs were cloned and partially sequenced. Inverse PCR was then employed to clone the corresponding region of the genomes of both the Jungle Fowl (JF) and White Leghorn (WL) parental DNA templates. Our results demonstrate that some of the CR1-PCR-generated DNAs arise from priming at an endogenous CR1 element, whereas others are due to chance complementarity between the CR1-PCR primer in use and random annealing sites in the genome, unrelated to a demonstrable CR1 element. In all five instances, it was possible to identify the sequence difference between the JF and WL parental DNAs that gave rise to the initial polymorphism and design allele-specific PCR primer sets that uniquely detect that polymorphism. In four of the five instances, the polymorphism was a one or two basepair sequence difference within the primer annealing site, but in the fifth case the responsible difference was outside, but very close to, the annealing site. In all instances the allele-specific PCR for the sequence polymorphism mapped identically with the corresponding CR1-PCR amplification polymorphism. We conclude that CR1-PCR provides an efficient and reliable mechanism for genome mapping in avians that can correlate linkage and physical mapping approaches.

Animals↗
PubMed2005

The latitudinal cline in the In(3R)Payne inversion polymorphism has shifted in the last 20 years in Australian Drosophila melanogaster populations.

Clinal variation has been described in a number of inversions in Drosophila but these clines are often characterized by cytological techniques using small sample sizes, and associations with specific genes are rarely considered. Here we have developed a molecular assay for In(3R)Payne in Drosophila melanogaster from eastern Australia populations. It shows in repeated samples that the inversion cline is very tightly associated with latitude and is almost fixed in tropical populations while relatively rare in temperate populations. This steep cline has shifted in position in the last 20 years. The heat shock gene, hsr-omega, located centrally inside the inversion sequence, shows a different clinal pattern to In(3R)Payne. These results suggest strong ongoing selection on In(3R)Payne over the last 100 years since the colonization of Australia that is partly independent of hsr-omega.

Animals↗
PubMed1995

Acute subarachnoid hemorrhage: MR imaging with fluid-attenuated inversion recovery pulse sequences.

PURPOSE: To evaluate the usefulness of fluid-attenuated inversion recovery (FLAIR) magnetic resonance (MR) imaging sequences in the detection of acute subarachnoid hemorrhage (SAH). MATERIALS AND METHODS: MR imaging with FLAIR sequences was performed with a 0.5-T superconducting unit in 20 patients (aged 30-72 years) with acute SAH due to a ruptured aneurysm and in 27 control subjects (aged 32-72 years). FLAIR images were obtained 2 hours to 2 days after ictus. Findings were evaluated and compared with computed tomographic (CT) findings. RESULTS: In all patients, acute SAH was clearly demonstrated as an area with signal intensity that was high relative to that of the normal cerebrospinal fluid and surrounding brain parenchyma at FLAIR imaging. This sequence was especially useful in demonstration of acute SAH in the posterior fossa, which was difficult to show at CT because of beam-hardening artifacts. In a double-blind comparison, no FLAIR images acquired in control subjects were confused with those acquired in control subjects were confused with those acquired in patients. CONCLUSION: FLAIR sequences reliably provide diagnostic images in patients with acute SAH.

Acute Disease↗