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Mathematical model to predict regions of chromatin attachment to the nuclear matrix.

The potentiation and subsequent initiation of transcription are complex biological phenomena. The region of attachment of the chromatin fiber to the nuclear matrix, known as the matrix attachment region or scaffold attachment region (MAR or SAR), are thought to be requisite for the transcriptional regulation of the eukaryotic genome. As expressed sequences should be contained in these regions, it becomes significant to answer the following question: can these regions be identified from the primary sequence data alone and subsequently used as markers for expressed sequences? This paper represents an effort toward achieving this goal and describes a mathematical model for the detection of MARs. The location of matrix associated regions has been linked to a variety of sequence patterns. Consequently, a list of these patterns is compiled and represented as a set of decision rules using an AND-OR formulation. The DNA sequence was then searched for the presence of these patterns and a statistical significance was associated with the frequency of occurrence of the various patterns. Subsequently, a mathematical potential value,MAR-Potential, was assigned to a sequence region as the inverse proportion to the probability that the observed pattern population occurred at random. Such a MAR detection process was applied to the analysis of a variety of known MAR containing sequences. Regions of matrix association predicted by the software essentially correspond to those determined experimentally. The human T-cell receptor and the DNA sequence from the Drosophila bithorax region were also analyzed. This demonstrates the usefulness of the approach described as a means to direct experimental resources.

Base Sequence↗

Transendocardial delivery of extracellular myocardial markers by using combination X-ray/MR fluoroscopic guidance: feasibility study in dogs.

PURPOSE: To demonstrate the feasibility of using a combination of x-ray fluoroscopic and magnetic resonance (MR) fluoroscopic (ie, x-ray/MR fluoroscopy) guidance for left ventricular (LV) catheterization and transendocardial delivery of extracellular tissue markers. MATERIALS AND METHODS: Experiments were performed in six dogs by using an x-ray/MR fluoroscopy system. The arterial guide wire and catheter were advanced into the heart with x-ray fluoroscopic guidance. The dogs were injected with 0.5, 1.0, and 2.0 mL of iohexol. For passive catheter tracking, a steady-state free precession MR imaging sequence was used. A steerable dual-lumen catheter was used to transendocardially inject a mixture of gadodiamide (0.05 mol/L) plus Evans blue dye (3%). An electrocardiographically gated dual-inversion-recovery MR imaging sequence was used to visualize the myocardial delivery of the gadodiamide-blue dye mixture. A high concentration of gadodiamide (0.5 mol/L) was used to demarcate the borders of the area of interest, or "hit the target." Blood pressure, heart rate, and oxygen saturation were measured before and after the intervention. Analysis of variance, Scheffé, and paired Student t tests were used for data analysis. RESULTS: LV catheterization via arterial access was feasible with two-dimensional x-ray fluoroscopic and three-dimensional MR fluoroscopic guidance. Delivery of the gadodiamide-blue dye mixture and the consequences of the procedure were monitored with MR imaging. Gadolinium-enhanced regions were bright on T1-weighted MR images, but they varied in size as a function of injectant volume. The mean sizes of these regions were 1.5% +/- 0.6 of the LV after the 0.5-mL injection of the mixture and 7.0% +/- 0.5 of the LV after the 2.0-mL injection (P <.001, Scheffé test). The corresponding mean sizes of the blue dye-enhanced regions were 2.3% +/- 0.6 and 8.3% +/- 0.4, respectively (P <.001). A high concentration of gadodiamide caused signal intensity loss around the gadolinium-enhanced regions. CONCLUSION: Transendocardial delivery of potential therapeutic solutions is feasible with x-ray/MR fluoroscopic guidance. The injection catheter can be navigated with MR imaging guidance to hit the target.

Animals↗

The pentanucleotide ATTGG, the "inverted CCAAT," is an essential element for HLA class I gene transcription.

The HLA class I genes, HLA-A, -B, and -C, contain an inverted CCAAT sequence (ATTGG) located 20 bp upstream of the canonical CCAAT and approximately 70 bp upstream of the transcription initiation site. We have investigated the transcriptional function of the class I inverted CCAAT sequence using the HLA-normal cell line, HeLa. Deletion, mutation, or inversion of the inverted CCAAT sequence abrogated or reduced the activity of the class I promoter, as assessed by luciferase reporter gene assays in transient gene expression experiments. This activity coincided with occupancy of the inverted CCAAT motif, as tested by electrophoretic mobility shift assays using the wild-type sequence and mutated variants of the sequence. The ATTGG-binding protein was not CP2, NF-1, or other known CCAAT-binding proteins, but the complex may contain a CP1/NF-Y-like protein. Our results indicate that this inverted CCAAT sequence is an essential element for the expression of HLA class I genes and that its transcriptional activity depends upon the sequence, position, and orientation of the pentanucleotide.

Base Sequence↗

Enthalpy/entropy compensation: influence of DNA flanking sequence on the binding of 7-amino actinomycin D to its primary binding site in short DNA duplexes.

The effect of the context of the flanking sequence on ligand binding to DNA oligonucleotides that contain consensus binding sites was investigated for the binding of the intercalator 7-amino actinomycin D. Seven self-complementary DNA oligomers each containing a centrally located primary binding site, 5'-A-G-C-T-3', flanked on either side by the sequences (AT)(n) or (AA)(n) (with n = 2, 3, 4) and AA(AT)(2), were studied. For different flanking sequences, (AA)(n)-series or (AT)(n)-series, differential fluorescence enhancements of the ligand due to binding were observed. Thermodynamic studies indicated that the flanking sequences not only affected DNA stability and secondary structure but also modulated ligand binding to the primary binding site. The magnitude of the ligand binding affinity to the primary site was inversely related to the sequence dependent stability. The enthalpy of ligand binding was directly measured by isothermal titration calorimetry, and this made it possible to parse the binding free energy into its energetic and entropic terms. Our results reveal a pronounced enthalpy-entropy compensation for 7-amino actinomycin D binding to this family of oligonucleotides and suggest that the DNA sequences flanking the primary binding site can strongly influence ligand recognition of specific sites on target DNA molecules.

Antineoplastic Agents↗

Constraints in chromosomal inversions in Escherichia coli are not explained by replication pausing at inverted terminator-like sequences.

Regions close to the replication terminus of the Escherichia coli chromosome are strongly refractory to genomic inversions. Since these regions also harbour polar replication terminator-like sequences or pause sites, we have investigated the possibility that slowing of replication as a result of pausing at inverted pause sites is responsible for inability to isolate stable inversions affecting these regions. A mutation in the tus gene is known to abolish replication pausing at terminators. We show here that the distribution of invertible and noninvertible segments along the chromosome is not affected by tus mutations. This observation eliminates replication pausing as a cause for the reduced fitness of bacteria harbouring certain chromosomal inversions.

Chromosome Inversion↗

The structure and population genetics of the breakpoints associated with the cosmopolitan chromosomal inversion In(3R)Payne in Drosophila melanogaster.

We report here the breakpoint structure and sequences of the Drosophila melanogaster cosmopolitan chromosomal inversion In(3R)P. Combining in situ hybridization to polytene chromosomes and long-range PCR, we have identified and sequenced the distal and proximal breakpoints. The breakpoints are not simple cut-and-paste structures; gene fragments and small duplications of DNA are associated with both breaks. The distal breakpoint breaks the tolkin (tok) gene and the proximal breakpoint breaks CG31279 and the tolloid (tld) gene. Functional copies of all three genes are found at the opposite breakpoints. We sequenced a representative sample of standard (St) and In(3R)P karyotypes for a 2-kb portion of the tok gene, as well as the same 2 kb from the pseudogene tok fragment found at the distal breakpoint of In(3R)P chromosomes. The tok gene in St arrangements possesses levels of polymorphism typical of D. melanogaster genes. The functional tok gene associated with In(3R)P shows little polymorphism. Numerous single-base changes, as well as deletions and duplications, are associated with the truncated copy of tok. The overall pattern of polymorphism is consistent with a recent origin of In(3R)P, on the order of Ne generations. The identification of these breakpoint sequences permits a simple PCR-based screen for In(3R)P.

Animals↗

Induction of an altered DNA conformation by an inversion rearrangement in the 5'-flanking DNA of a U1 RNA gene.

The anomalous electrophoretic behavior of a 686 base pair restriction fragment containing an in vitro-generated inversion mutation within the enhancer region of a chicken U1 RNA gene was investigated. This DNA fragment migrated with an abnormally slow mobility in polyacrylamide gels but migrated normally in agarose gels relative to the wild type fragment of identical size and base composition. In polyacrylamide gels, the degree of retardation was enhanced at low temperature, a phenomenon associated with bent DNA. A putative site of bending was localized at or near one end of the inverted region. These data suggest that the altered DNA conformation results from the juxtaposition of two normally remote DNA sequences.

Base Sequence↗

Molecular cloning, sequencing and characterization of omp48, the gene encoding for an antigenic outer membrane protein from Aeromonas veronii.

AIMS: To clone, sequence and characterize the gene encoding the Omp48, a major outer membrane protein from Aeromonas veronii. METHODS AND RESULTS: A genomic library of Aer. veronii was constructed and screened to detect omp48 gene sequences, but no positive clones were identified, even under low stringency conditions. The cloned gene probably was toxic to the host Escherichia coli strain, so the cloning of omp48 was achieved by inverse PCR. The nucleotide sequence of omp48 consisted of an open reading frame of 1278 base pairs. The predicted primary protein is composed of 426 amino acids, with a 25-amino-acid signal peptide and common Ala-X-Ala cleavage site. The mature protein is composed of 401 amino acids with a molecular mass of 44,256 Da. CONCLUSIONS: The omp48 gene from Aer. veronii was cloned, sequenced and characterized in detail. BLAST analysis of Omp48 protein showed sequence similarity (over 50%) to the LamB porin family from other pathogenic Gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial diseases are a major economic problem for the fish farming industry. Outer membrane proteins are potentially important vaccine components. The characterization of omp48 gene will allow further investigation of the potential of Omp48 as recombinant or DNA vaccine component to prevent Aer. veronii and related species infections in reared fish.

Aeromonas↗

Molecular cloning, sequence, and expression of cynomolgus monkey cholesteryl ester transfer protein. Inverse correlation between hepatic cholesteryl ester transfer protein mRNA levels and plasma high density lipoprotein levels.

A cDNA clone containing the coding region for cynomolgus monkey cholesteryl ester transfer protein (CETP) was isolated by the polymerase chain reaction with primers based on the human CETP cDNA sequence and cDNA synthesized from liver poly (A+) RNA. Analysis of that cDNA indicated that the nucleotide and amino acid sequences of cynomolgus monkey CETP were greater than 95% homologous with the human sequences. A fragment of the cDNA was used to develop an internal-standard/RNAse protection assay that allowed precise quantification of CETP mRNA levels. Analysis of total RNA from various tissues with this assay revealed that the liver and thoracic aorta expressed high levels of CETP mRNA; the mesenteric fat, adrenal gland, spleen, and abdominal aorta had low but detectable levels of the mRNA; and the brain, kidney, intestine, and skeletal muscle had undetectable levels of that mRNA. When the monkeys were made hypercholesterolemic by a high-fat, high-cholesterol (HFHC) diet, hepatic levels of CETP mRNA increased from 1.6 +/- 0.4 pg/micrograms total RNA (mean +/- SEM) to 4.1 +/- 0.8 pg/micrograms (p less than 0.005); mesenteric fat CETP mRNA increased from 0.4 +/- 0.1 pg/micrograms total RNA to 5.3 +/- 2.2 pg/micrograms (p less than 0.05); and plasma CET activity increased approximately fourfold. The CETP mRNA levels in the thoracic and abdominal aortas were not significantly increased in monkeys fed the HFHC diet, even though those animals had gross atherosclerosis. The apoprotein E mRNA levels, however, were markedly increased in the aortas of monkeys with atherosclerosis, with the largest increase occurring in the abdominal aorta. Taken together, these data suggest that lipid deposition in the artery was not accompanied by increased expression of the CETP gene in that tissue. Statistical analysis showed that a strong, negative correlation existed between hepatic CETP mRNA levels and both high density lipoprotein cholesterol (r = -0.85, p less than 0.001) and apoprotein A-I (r = -0.84, p less than 0.001). These data suggest that HFHC diet-induced changes in high density lipoprotein metabolism may be linked to altered expression of a function CETP gene.

Animals↗

Molecular characterization of a myelodysplasia-associated chromosome 7 inversion.

Chromosome 7 abnormalities are observed in a wide range of myeloid disorders, particularly myelodysplasia (MDS) and acute myeloid leukaemia (AML). Monosomy 7 and 7q deletions are the most frequent abnormalities, although translocations and inversions involving 7q also occur. The region 7q22--q34 may contain as many as four distinct minimal regions of deletion (MDRs), which are thought to contain one or more myeloid tumour-suppressor genes. We have defined previously the proximal breakpoint of a constitutional 7q22--q34 inversion, carried in a cell line derived from a member of a family with a history of MDS. A YAC clone spanning this breakpoint was identified. Both inversion breakpoints have now been cloned and sequenced, placing the proximal breakpoint 40 kb centromeric to the TAC2 (tachykinin 2) gene and the distal breakpoint 42 kb telomeric to the SSBP (mitochondrial single-stranded DNA-binding protein) gene. Sequence alignments revealed small (3--4 bp) duplications at the inversion breakpoints, suggesting that the mechanism of inversion involved the creation of staggered breaks and filling in of the overhanging ends. A 190-bp Alu--Alu deletion close to the distal breakpoint was also detected and may have contributed to the inversion.

Autoradiography↗

Microsatellite and mitochondrial genetic differentiation of Anopheles arabiensis (Diptera: Culicidae) from western Kenya, the Great Rift Valley, and coastal Kenya.

The population genetic structure of the African malaria vector Anopheles arabiensis from western Kenya, the Great Rift Valley, and coastal Kenya was investigated using 12 microsatellite loci and a partial sequence of mtDNA dehydrogenase gene subunit 5 (ND5). The mean number of alleles and the observed heterozygosity were similar for the mosquito populations from the three regions as revealed by the microsatellite data. A total of 30 polymorphic sites in the ND5 gene defined 39 haplotypes. Six haplotypes were shared among four populations from the three distinct ecological conditions, and they constituted 92% of the total number of individuals sequenced. Mitochondrial haplotype and nucleotide diversity were high. Microsatellite markers within polymorphic inversions revealed a level of genetic differentiation (F(ST) = 0.116) four to seven times higher than markers outside inversions (F(ST) = 0.016) or inside fixed inversions (F(ST) = 0.027). Mitochondrial ND5 gene sequences did not reveal significant genetic differentiation for the same four populations (phi(ST) = -0.008). The contrasts in the level of genetic differentiation between microsatellite markers inside polymorphic inversions, the mitochondrial ND5 gene, and microsatellite markers outside inversions suggest that the level of genetic differentiation in An. arabiensis populations across the Great Rift Valley varies significantly among different areas of the genome. Variations in the degree of genetic differentiation with respect to the chromosomal location of microsatellite markers may result from intrinsic characteristics of the markers, demographic or historic factors affecting these populations, and the possible adaptive significance of chromosomal inversions to climatic conditions.

Animals↗

Genomic organization of the human T-cell antigen-receptor alpha/delta locus.

Two clusters of overlapping cosmid clones comprising about 100 kilobases (kb) at the human T-cell antigen-receptor alpha/delta locus were isolated from a genomic library. The structure of the germ-line V delta 1 variable gene segment was determined. V delta 1 is located 8.5 kb downstream of the V alpha 13.1 gene segment, and both V segments are arranged in the same transcriptional orientation. The V alpha 17.1 segment is located between V delta 1 and the D delta, J delta, C delta region (containing the diversity, joining, and constant gene segments). Thus, V delta and V alpha segments are interspersed along the chromosome. The germ-line organization of the D delta 2, J delta 1, and J delta 2 segments was determined. Linkage of C delta to the J alpha region was established by identification of J alpha segments within 20 kb downstream of C delta. The organization of the locus was also analyzed by field-inversion gel electrophoresis. The unrearranged V delta 1 and D delta, J delta, C delta regions are quite distant from each other, apparently separated by a minimum of 175-180 kb.

Base Sequence↗

Development of a deer mouse whole-genome radiation hybrid panel and comparative mapping of Mus chromosome 11 loci.

A 5000-rad whole-genome radiation hybrid cell panel (BW5000) was developed for mapping the deer mouse (Peromyscus maniculatus bairdii) genome. The panel consists of 103 cell lines and has an estimated marker retention frequency of 63.9% (range, 28%-88%) based on PCR typing of 30 Type I (coding gene) and 25 Type II (microsatellite) markers. Using the composite Mus map, Type I markers were selected from six Mus chromosomes, 22 of which are on Mus Chr 11. Fifteen of the Mus Chr 11 markers were simultaneously mapped on an interspecific (P. maniculatus x P. polionotus) backcross panel to test the utility of the radiation hybrid panel, create a framework map, and help establish gene order. The radiation hybrids have effectively detected linkage in the deer mouse genome between markers as far apart as 6.7 cM and resolved markers that are, in the Mus genome, as close as 0.2 Mb. Combined results from both panels have indicated a high degree of gene order conservation of the telomeric 64 cM of Mus Chr 11 in the deer mouse genome. The remaining centromeric portion also shows gene order conservation with the deer mouse but as a separate linkage group. This indicates a translocation of that portion of Mus Chr 11 in P. maniculatus and is consistent with rearrangement breakpoints observed between Mus and other mammalian genomes, including rat and human. Furthermore, this separate linkage group is likely to reside in a chromosomal region of inversion polymorphism between P. maniculatus and P. polionotus.

Animals↗

Heart rate variability and spontaneous baroreflex sequences: implications for autonomic monitoring during hemorrhage.

BACKGROUND: Noninvasive procedures for predicting progression to hemodynamic instability during induced central hypovolemia in humans were evaluated. The purpose of this study was twofold: (1) to track changes in autonomic function induced by a model of hemorrhage, and (2) to determine whether measures of autonomic function are reliable without strict control of breathing. METHODS: Electrocardiogram, respiratory frequency, and arterial pressure during progressive lower body negative pressure were recorded for 10 subjects, and for a separate sample of 20 subjects during 5-minute periods of spontaneous breathing or controlled-frequency breathing at 15 breaths per minute.. Heart rate variability was calculated in both time and frequency domains. Up and down baroreflex sequences were calculated with linear regression analysis between sequential changes in systolic pressures and accompanying parallel changes in R-R intervals. RESULTS: Heart rate variability (r = 0.92) and up (r = 0.90) and down (r = 0.96) sequences changed in direct inverse relation to decreased central volume as produced by progressive increases in lower body negative pressure, whereas mean arterial pressures remained constant (r = 0.26). Neither heart rate variability nor up and down baroreflex sequences were affected by the mode of breathing. CONCLUSIONS: Analysis of heart rate variability and baroreflex sequences in hemorrhaging patients may provide advance recognition of those at risk for progression to shock. In conjunction with more traditional modes of assessing volume status, tracking early changes in autonomic function may improve resuscitation efforts for the hemodynamic compromised patient.

Adolescent↗

Exchange of spacer regions between rRNA operons in Escherichia coli.

The Escherichia coli rRNA operons each have one of two types of spacer separating the 16S and 23S coding regions. The spacers of four operons encode tRNA(Glu2) and the other three encode both tRNA(Ile) and tRNA(Ala1B). We have prepared a series of mutants in which the spacer region of a particular rrn operon has been replaced by the opposite type. Included among these were a mutant retaining only a single copy of the tRNA(Glu2) spacer (at rrnG) and another retaining only a single copy of the tRNA(Ile)-tRNA(Ala1B) spacer (at rrnA). While both mutants grew more slowly than controls, the mutant deficient in tRNA(Glu2) spacers was more severely affected. At a frequency of 6 X 10(-5), these mutants phenotypically reverted to faster growing types by increasing the copy number of the deficient spacer. In most of these phenotypic revertants, the deficient spacer type appeared in a rrn operon which previously contained the surplus type, bringing the ratio of spacer types closer to normal. In a few cases, these spacer changes were accompanied by an inversion of the chromosomal material between the donor and recipient rrn operons. Two examples of inversion of one-half of the E. coli chromosome between rrnG and rrnH were observed. The correlation of spacer change with inversion indicated that, in these particular cases, the change was due to an intrachromatid gene conversion event accompanied by a reciprocal crossover rather than reciprocal exchange between sister chromatids.

Base Sequence↗

Transposon Tn951 (TnLac) is defective and related to Tn3.

Tn951 is flanked by two perfect inverted repeats of 41 bp which include the 38 bp sequence of the IR of Tn3. Tn951 also contains the last 100 bp of the tnpA gene but with at least two mutations. However, beyond nucleotide 137 the sequences diverge and hybridization experiments show that Tn951 lacks at least the first two thirds of the tnpA gene. In agreement with these observations Tn951 does not transpose by itself at a detectable frequency but can be complemented by the tnpA gene of Tn801 or Tn3. Tn501, Tn1721 and gamma delta do not complement Tn951 transposition. Transposition of Tn951 duplicates 5 bp of target DNA sequence.

Base Sequence↗

Hin-mediated site-specific recombination requires two 26 bp recombination sites and a 60 bp recombinational enhancer.

The alternate expression of flagellin genes in Salmonella is the result of an inversion of a 996 bp segment of chromosomal DNA. We have analyzed the components of this site-specific recombination reaction in an in vitro system derived from E. coli. Efficient Hin-mediated inversion requires the 20,000 MW Hin protein and a proteinase K-sensitive host component. The supercoiled DNA substrate must contain two 26 bp recombination sites in inverted configuration and a 60 bp sequence that increases the rate of recombination over 20-fold. This recombinational enhancer can function at many different locations and consists of at least two noncontiguous sequence domains whose relative orientation, but not precise spacing, with respect to each other is important. Synthetically derived wild-type and mutant recombination sites were constructed to analyze the sequence and structural features that are important within the recombination site.

Bacterial Proteins↗