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PubMed1997

Temporal processing disorders in patients with Broca's aphasia.

We report an association between a language deficit following brain lesion and a new strategy in temporal integration. Patients with different brain lesions mentally grouped sequences of identical acoustic stimuli generated at various frequencies. They were asked while listening to the stimuli to accentuate every second, third or other stimulus to create an individual rhythmic pattern. After each sequence patients reported how many stimuli they had united into a perceptual unit. The integration interval was defined as the number of reported stimuli multiplied by the temporal interval between two successive stimuli. Results indicate different integration strategies depending on the lesion site, i.e. Broca's aphasics behaving differently than all other patient groups. At lower frequencies they showed longer, at higher frequencies they displayed shorter integration. From this observation we conclude that the Broca's patients acquired a new strategy because of the lesion; they relied on mental counting and less on automatic temporal integration, which is usually the case.

Adult↗
PubMed1999

[Incidence of extended spectrum beta lactamase in Escherichia coli in a university hospital from 1994-1996].

BACKGROUND: The aim of this study was to evaluate the incidence of TEM- and SHV-type extended spectrum betalactamases (ESBLs) in Escherichia coli in a 700-bed teaching hospital between 1994 and 1996. MATERIAL AND METHODS: Strains that presented reduced diameters to third-generation cephalosporins, as identified by disc diffusion techniques, were studied. The betalactamases involved were characterized by determination of the isoelectric point, hydrolysis profile, gene detection by polymerase chain reaction (PCR), and sequencing of the amplified products. RESULTS: 96 strains (1.4%) out of 7,054 strains of E. coli isolated between 1994 and 1996 showed decreased susceptibility to third-generation cephalosporins and only 4 strains (0.06%) produced ESBLs. Two strains produced SHV-2 and two produced TEM-12. CONCLUSIONS: The contribution of ESBL production to resistance to third-generation cephalosporins was low: 0.06% of all the E. coli strains isolated between 1994 and 1996.

Escherichia coli↗
PubMed1984

Mechanisms for the generation of src-deletion mutants and recovered sarcoma viruses: identification of viral sequences involved in src deletions and in recombination with c-src sequences.

The precise src deletions in six transformation-defective (td) deletion mutants derived from the Schmidt-Ruppin strain of Rous sarcoma virus were determined by sequence analysis. Examination of the parental viral sequences neighboring the junctions of deletions in these td mutants revealed that these regions contained either directly repeated or inverted complementary sequences ranging from 9 to 28 nucleotides. Five td mutants were found to contain deletions flanked by directly repeated sequences, of which the 3' direct repeat was retained whereas the 5' direct repeat was deleted in the resulting td viral RNA. In the deletions of two td mutants where inverted complementary sequences were present at junctions of the deletions, both copies of the inverted complementary sequence were deleted in the td viral RNA. It is proposed from these observations that deletions of these mutants have been generated during the synthesis of minus-strand viral DNA by reverse transcriptase by jumping over a sequence flanked by direct repeats or by skipping a stem-and-loop structure formed via inverted complementary sequences on the viral RNA template. Data provide further information on the sequences in the td viral genome that are required for the generation of recovered sarcoma viruses (rASVs) by recombination with c-src. Sequence data of td viruses revealed that retaining as few as 82 nucleotides of the 3' src coding sequence is sufficient, whereas retaining as much as one-third of the 5' src but none of the 3' src coding sequences is not sufficient, for the generation of rASVs. Those that generate replication-competent rASVs retain, in addition to the 3' src region, a portion of the 5' src and/or its immediate upstream sequence that is homologous to exon 1 of the c-src DNA. These two sequence domains apparently provided 5' and 3' homologous regions for recombination between td viral genome and c-src DNA resulting in nondefective rASVs. Td109, which was shown previously to generate only replication-defective rASVs, retains 296 nucleotides of the 3' src sequence but lacks all the 5' src and 316 nucleotides of its immediate upstream region. It is concluded that the 5' src coding sequence and its immediate upstream region are not essential for the generation of rASVs. However, retaining a portion of those sequences is required for the generation of replication-competent rASVs.

Animals↗
PubMed1987

Structure and expression of pea mitochondrial F1ATPase alpha-subunit gene and its pseudogene involved in homologous recombination.

We have characterized four pea mitochondrial DNA segments carrying the F1ATPase alpha-subunit coding sequences. These four types share a common 1.7-kb repeat sequence flanked by four combinations of two different left- and right-hand sequences. These results suggest that the alpha-subunit genes locate at the homologous recombination sites in the pea mitochondrial genome and that homologous recombination between two of these loci generates the other two types of structures. The uninterrupted alpha-subunit coding sequence of 1,521 bp is present in two of these loci. A rearrangement of 965 bp 3' to the ATG initiation codon generates two copies of pseudogenes where the C-terminal two-thirds of the alpha-subunit coding sequence is replaced with an unidentified coding frame. The other border of sequence divergence is located 733 bp upstream of the ATG initiation codon. Although multiple forms of alpha-subunit gene transcripts are present in mitochondria, the pseudogenes do not seem to express any alpha-subunit-related polypeptide.

Amino Acid Sequence↗
PubMed1997

A minichromosome carrying a pigmentation gene and brook trout DNA sequences in transgenic rainbow trout.

We describe the transmission of an introduced minichromosome of brook trout (Salvelinus fontinalis) origin, carrying a pigmentation gene, through three generations in rainbow trout (Oncorhynchus mykiss). The minichromosome was originally introduced into gynogenetic albino rainbow trout using gamma-irradiated brook trout sperm. In the third generation, the presence of the minichromosome was correlated with pigmentation. A brook trout specific interspersed repeat DNA sequence, Fok I, was also correlated with pigmentation in these individuals. This system, the first clearly documented example of induced chromosome mediated gene transfer at the organismal level, could have applications in studies of gene mapping, development, gene regulation, and chromosome function.

Animals↗
PubMed1993

Response of the 23Na-NMR double-quantum filtered signal to changes in Na+ ion concentration in model biological solutions and human erythrocytes.

Double quantum filtered (DQF) 23Na-NMR signals were evaluated as a function of [Na+] at constant temperature in two model systems (bovine serum albumin (BSA) and Ficoll 400) and in human red blood cells (RBCs). In model systems, the ratio of double quantum filtered to single quantum (SQ) signal intensities was independent of [Na+], even over a wide range of Na+/K+ ratios. Varying the DQF preparation time affected only the DQF signal intensity. In contrast, in human red blood cells (RBCs) the shape and phase of the DQF intracellular Na+ signal (Na+in) varied as a function of preparation time. Similar observations in cartilage [Eliav, U., Shinar, H. and Navon, G. (1992) J. Magn. Reson. 28, 223-229] have been attributed to the generation of a second- and a third-rank tensor by the DQF pulse sequence, resulting from Na+ ion ordering. By using a DQF sequence which isolates the second-rank tensor only, this component was found to originate from the intracellular Na+ ion pool in human RBCs, as well as from interactions of Na+ ions with the extracellular face of the plasma membrane. The residual quadrupolar splitting for the signal originating from the former environment was shown to be less than the SQ linewidth, explaining its absence in SQ spectra, and this was confirmed by two-dimensional DQF 23Na-NMR experiments. By isolating the contribution from the third-rank tensor exclusively, the ratio of DQF:SQ signal intensities for Na+in in human RBCs was shown to be constant over a 4-fold change in [Na+in] produced by addition of an ionophore (nystatin). This indicates that such changes in physiological state do not alter the efficiency of DQF signal generation in human RBCs.

Animals↗
PubMed2000

MHC diversity in Caucasians, investigated using highly heterogeneous noncoding sequence motifs at the DQB1 locus including a retroviral long terminal repeat element, and its comparison to nonhuman primate homologues.

Long terminal repeats (LTRs) are common retrovirus-related sequences spread throughout the human genome. We previously reported the human-specific integration of one LTR (DQLTR3) located 15 kb upstream of HLA DQB1. To elucidate the contribution of retroviral sequences to the variability and phylogenetic background of HLA DQB1 we investigated another LTR (DQLTR13), located 1.3 kb upstream of HLA DQB1, in German families, great apes, and Old World monkeys. Within German families, DQLTR13 presence was strongly linked to HLA DQB1*0302, *0303, and *0402 haplotypes. All other haplotypes had a low frequency or were devoid of DQLTR13. Phylogenetic analysis of DQLTR13 and adjacent nucleotide sequences in humans and non-human primates revealed a high degree of similarity and recent origin of HLA DQB1*0302, *0303, and *0402. Nevertheless, two lineages leading to DQB1*0301 and *0302 were generated by an ancient split of a DQB1*0301, *0302 progenitor. A third lineage consisting of DQB1*05/*06-related sequences may have evolved from the DQB1*0302 lineage, and a DQB1*0201-related sequence shared common ancestry with DQB1*0301. Among the human haplotypes, HLA DQB1*0201 and *0301 are linked to two different DQA1 alleles. Based on the small genetic distance of DQLTR13 as well as the adjacent sequences on these haplotypes, we suggest that a recent recombination is responsible for these associations. In the analysis of nonhuman primate species, we detected DQLTR13 in two lowland gorillas, dating the integration at at least 8 million years ago. We therefore conclude that noncoding sequences up to 1.3 kb upstream of DQB1 provide novel insight into the generation of MHC gene diversity.

Animals↗
PubMed1990

The Arabidopsis thaliana plasma membrane H(+)-ATPase multigene family. Genomic sequence and expression of a third isoform.

The plasma membrane of higher plants contains a H(+)-ATPase as its major ion pump. This enzyme belongs to the P-type family of cation-translocating enzymes and generates the proton-motive force that drives solute uptake across the plasma membrane. In Arabidopsis thaliana the plasma membrane H(+)-ATPase is encoded by a multigene family (Harper, J. F., Surowy, T. K., and Sussman, M. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1234-1238). The complete genomic sequence of a third Arabidopsis H(+)-ATPase isoform (referred to as AHA2) is presented here, and the predicted protein sequence is compared with previously published AHA1, AHA3, and tobacco Nicotiana plumbaginifolia NP1 isoforms. The AHA2 gene is most similar to AHA1, with predicted proteins containing 95% amino acid identity. The mRNA start site and 5'-untranslated sequence for AHA2 were determined from cDNA amplified by the polymerase chain reaction. The 5' region contains a 23-base pair (bp) polypyrimidine sequence and a short upstream reading frame. In comparison with the 16 introns reported in AHA3, AHA2 is missing one intron in the 5'-untranslated region and a second intron in the C-terminal coding region. An unusually large intron for Arabidopsis (greater than 1000 bp) is present at the beginning of the coding sequence of both AHA2 and AHA3. In the 3'-untranslated sequence of AHA1 and AHA2 but not AHA3, there is a 65-bp region of 85% identity and a second shorter region of 16-bp identity harboring an unusual putative poly(A) addition signal (dTTTGAAGAAACAAGGC). Northern blot analysis indicates that AHA2 mRNA relative to total cellular RNA is expressed at significantly higher levels in root tissue as compared with shoot tissue.

Amino Acid Sequence↗
PubMed1992

Close amino acid sequence relationship between the new plasmid-mediated extended-spectrum beta-lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca.

Isolated from an Escherichia coli strain MEN-1 is a plasmid-mediated beta-lactamase that confers resistance to methoxy imino third-generation cephalosporins. The protein purified to homogeneity was digested by trypsin, chymotrypsin and endoproteinase Asp-N. Amino acid sequence determinations of the resulting peptides gave rise to the alignment of the 263 residues of the beta-lactamase. From amino acid sequence comparison MEN-1 was found to share more than 72% identity with the chromosomally mediated beta-lactamases of Klebsiella oxytoca. Therefore, MEN-1 is the first transferable extended-spectrum beta-lactamase which is not directly derived from the widespread TEMs or SHV-1 penicillinases with which it presents less than 39% identity.

Amino Acid Sequence↗
PubMed2001

Diagnosis of Liddle syndrome by genetic analysis of beta and gamma subunits of epithelial sodium channel--a report of five affected family members.

OBJECTIVE: To screen the gene mutation in beta and gamma subunits of the epithelial sodium channel (ENaC) of a Chinese family, some of whose members are clinically diagnosed as suffering from Liddle syndrome. METHODS: Twelve family members were recruited to the study. Among them, two brothers had been clinically diagnosed as suffering from Liddle syndrome. Peripheral blood samples were collected from all members of the family and total genomic DNA was prepared for genetic analysis. Polymerase chain reaction (PCR) was used for amplifying the last exon of beta (codon 513-673) and gamma (codon 503-632) subunits of the ENaC gene. PCR products were purified and subjected to a direct DNA sequence analysis. RESULTS: Genetic analysis of the beta ENaC gene revealed a missense mutation of CCC to CTC at codon 616 in four middle-aged men of the second generation and one young woman of the third generation. There was no mutation of the gamma ENaC gene in any of the individuals examined. CONCLUSION: Through direct DNA sequencing analysis, we diagnosed the disease present in five members of a Chinese family as Liddle syndrome, and excluded it in some other young offspring suffering from the monogenic disease. Our results provide further evidence that Pro616 is a critical amino acid that has a key role in the inhibition of sodium channel activity.

Adolescent↗
PubMed1984

A model for the mechanism of alkylation mutagenesis.

The phenomenology of mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine and related alkylating agents is reviewed and a three-step model for the molecular events of mutagenesis is presented. The first step is the production of miscoding lesions, especially O6-methylguanine, and the induction and synthesis of methyltransferase. The second step is the generation of DNA sequences in which O6-methylguanine is paired with thymine. The third step is the conversion of this abnormal base pair to an adenine-thymine pair completing the production of a transition mutation. At each of these steps, factors which affect the ultimate mutation frequency are outlined. The model is then described formally and the limits of the model are discussed.

Alkylating Agents↗
PubMed2000

Phylogeny of Bacillus sphaericus-like organisms.

The mesophilic round-spored bacteria embrace four species, namely Bacillus sphaericus, Bacillus fusiformis, Bacillus silvestris and Bacillus pasteurii. Although not displayed by all strains, mosquito pathogenicity is a noteworthy characteristic of B. sphaericus sensu lato. Phylogenetic analysis based on 16S rDNA sequences from 58 strains identified as B. sphaericus was used to examine the genetic heterogeneity of the taxon. Results from sequence analysis were compared with whole-cell fatty acid profiles and other phenotypic determinations. The B. sphaericus-like strains segregated into seven distinct clusters in a phylogenetic tree generated from 16S sequences. One cluster represented B. sphaericus and another B. fusiformis. A third cluster containing all of the pathogenic strains was closely related to, or was possibly part of, the B. fusiformis group. The remaining four groups were distinct and represented unnamed taxa that were more closely related to B. sphaericus and B. fusiformis than to the psychrophilic round-spored species, Bacillus globisporus and Bacillus psychrophilus. Groups based on phenotypic analysis corresponded to the 16S rDNA phylogenetic clusters. Data showed that B. sphaericus was genetically and phenotypically a highly heterogeneous taxon including at least seven genetically distinct taxa. The pathogenic strains were members of a distinct group and not of the species B. sphaericus sensu stricto. This heterogeneity partially accounts for the apparent variability of mosquito pathogenicity among B. sphaericus strains.

Animals↗
PubMed1998

Nucleotide sequence of the 3'-terminal two-thirds of the grapevine leafroll-associated virus-3 genome reveals a typical monopartite closterovirus.

The RNA genome of grapevine leafroll-associated closterovirus-3 (GLRaV-3) was cloned as a cDNA generated from GLRaV-3-specific dsRNA, and a partial genome sequence of 13154 nucleotides (nt) including the 3' terminus was determined. The sequenced portion contained 13 open reading frames (ORFs) potentially encoding, in the 5'-3' direction, proteins of > 77 kDa (ORF1a; helicase, HEL), 61 kDa (ORF1b; RNA-dependent RNA polymerase, RdRp), 6 kDa (ORF2), 5 kDa (ORF3, small transmembrane protein), 59 kDa (ORF4; heat shock protein 70, HSP70), 55 kDa (ORF5), 35 kDa (ORF6; coat protein, CP), 53 kDa (ORF7; diverged coat protein, CPd), 21 kDa (ORF8), 20 kDa (ORF9), 20 kDa (ORF10), 4 kDa (ORF11), 7 kDa (ORF12), and an untranslated region of 277 nt. ORF1b is probably expressed via a +1 ribosomal frameshift mechanism, most similar to that of lettuce infectious yellows virus (LIYV). Phylogenetic analysis using various gene sequences (HEL, RdRp, HSP70 and CP) clearly demonstrated that GLRaV-3, a mealybug-transmissible closterovirus, is positioned independently from aphid-transmissible monopartite closteroviruses (beet yellows, citrus tristeza and beet yellows stunt) and whitefly-transmissible bipartite closterovirus (lettuce infectious yellows, LIYV). However, another alleged mealybug-transmissible closterovirus, little cherry virus, was shown to be more closely related to the whitefly-transmissible LIYV than to GLRaV-3.

Amino Acid Sequence↗
PubMed1991

Mapping using unique sequences.

Theoretical predictions are given for the progress expected, when mapping DNA by identifying clones containing specific unique sequences. Progress is measured in three ways; however, all results depend on (dimensionless counterparts of) the number of clones and the number of unique sequences used. Furthermore, the effects of clone length dispersion are included in the theoretical predictions. Both the clones in the library and the unique sequences are assumed to be generated randomly, with uniform probability of originating at any base in the region to be mapped. The first measure of progress is the expected length fraction of the region to be mapped covered by at least one clone, when clones containing at least one unique sequence are included in the map. The second measure of progress is the expected length fraction of the region to be mapped in "covered intervals", an interval being the region between adjacent unique sequences. Alternative definitions for clones covering an interval are analyzed. The third measure of progress is the expected number of clone islands generated; an island covers successive intervals. Finally, using these measures of progress, we compare the efficiency of this new mapping strategy with conventional clone mapping strategies.

Chromosome Mapping↗
PubMed1997

Porcine aromatases: studies on tissue-specific, functionally distinct isozymes from a single gene?

Aromatase cytochrome P450 (P450arom) is expressed in a variety of tissues. Pigs express P450arom as bilaminar blastocysts in utero, and thereafter in the gonads, adrenal glands and placenta. Our studies also demonstrate the existence of porcine isozymes of P450arom which differ substantially in their amino acid composition and function. The placental isoform, most similar to P450arom in other mammals, consists of 503 amino acids. The ovarian isoform, expressed in both theca and granulosa cells, is a 501 amino acid protein exhibiting less than 20% of the activity of the placental isozyme. Furthermore, it is inhibited not only by CGS16949A but also by etomidate which does not inhibit the placental P450arom. Partial sequences generated by the rapid amplification of the cDNA ends (RACE) procedure indicate that the expression of a third isoform in the blastocyst is switched to the placental isozyme during differentiation of the fetal membranes. In addition, these transcripts, and others from the theca, granulosa, testes, adrenal glands and placenta demonstrate differences in the 5'-untranslated region (putative exon I) suggestive of tissue-specific alternative splicing. An identical 5'-untranslated sequence was obtained from transcripts expressed in the theca and granulosa. Testes and adrenal transcripts also have identical 5' ends, which differ substantially from the ovarian sequence. Blastocyst and placenta 5'-untranslated sequences differ from each other and from those expressed in the gonads and adrenals. Several tissue-specific transcripts thus encode porcine P450arom. Interestingly, distinct 5' sequences exist for ovarian and testes P450arom mRNAs, suggesting different promoters and therefore regulation in the male and female gonads. The molecular origins of the functional isoforms and the tissue-specific transcripts are uncertain, however partial genomic sequence and other genetic analyses suggest the existence of multiple genes. However, sequence alignment of the placental and ovarian isoforms indicates complete conservation of putative exon III, so that complex splicing remains a possibility. Clearly, the regulation of P450arom expression is more complex in the pig than in other vertebrates investigated to date.

Amino Acid Sequence↗
PubMed1995

Structural organization of the murine D3 dopamine receptor gene.

We have cloned the gene encoding the murine D3 dopamine receptor and have analyzed its intron-exon structural organization, to gain a better understanding of the detailed architecture of the D2 dopamine receptor genes. Restriction and sequence analysis reveal the presence of six introns, in contrast to the five introns previously reported for the rat D3 receptor. The extra intron is located in the receptor's putative third cytoplasmic loop and generates an intron-exon organization directly analogous to that found in the D2 receptor gene. In addition, we have sequenced the 5' and 3' nontranslated sequences flanking the coding region and have identified a putative poly(A) adenylation signal. These sequences are found to have a far lower homology with the corresponding rat nontranslated sequences than is found for the D2 receptor, suggesting that the control of D3 receptor expression may vary more between species than the control of D2 receptor expression.

Amino Acid Sequence↗
PubMed2000

Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmid-mediated CMY-4 and permeability alteration.

Klebsiella pneumoniae BM2974 isolated from an abdominal abcess was resistant to high concentrations of all available beta-lactams, including recently developed third-generation cephalosporins and carbapenems. Isoelectric focusing of beta-lactamases and amplification, cloning and sequencing of the corresponding genes, together with conjugation and transformation experiments, indicated that, in addition to the chromosomally encoded beta-lactamase, the strain produced three plasmid-mediated beta-lactamases with pIs of 5.4, 8.2 and 9.0, which corresponded to TEM-1, SHV-5 and AmpC-type CMY-4, respectively. Strain BM2974 also lacked a major outer membrane protein of c. 40 kDa which was present in the spontaneous imipenem-susceptible revertant BM2974-1. We suggest that imipenem resistance in strain BM2974 is attributable to production of CMY-4 beta-lactamase combined with permeability alteration.

Bacterial Outer Membrane Proteins↗