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Brain structure and task-specific increase in expression of the gene encoding syntaxin 1B during learning in the rat: a potential molecular marker for learning-induced synaptic plasticity in neural networks.

The mRNAs encoding the synaptic vesicle proteins syntaxin 1B and synapsin I were measured using in situ hybridization in several brain regions--the dentate gyrus, CA3 and CA1 of the hippocampus, the parietal, the motor and prefrontal cortices and the core and shell of the accumbens--of rats that were learning a spatial reference or working memory task on a radial arm maze. The mRNA encoding syntaxin 1B was significantly increased in all hippocampal regions in rats learning the working memory task, whereas it was increased in the prelimbic area of the prefrontal cortex and the shell of the accumbens in rats learning the spatial reference memory task. No change in mRNA encoding syntaxin 1B was observed in the motor and parietal and cortices or the core of the accumbens, and the mRNA encoding synapsin I was not significantly different from that of naive caged controls or rats running the maze for continuous reinforcement in any of the brain structures examined. These results demonstrate that the gene encoding a key member of synaptic vesicle function is up-regulated in a task- and brain-specific manner during learning. They are discussed in terms of the potential role this protein may play in trans-synaptic propagation of plasticity within specific neural networks as a function of the information required in the laying down of different types of memory.

Animals↗

Tetrodotoxin-resistant Na+ channels in human neuroblastoma cells are encoded by new variants of Nav1.5/SCN5A.

Both tetrodotoxin-sensitive (TTX-S) and TTX-resistant (TTX-R) voltage-dependent Na+ channels are expressed in the human neuroblastoma cell line NB-1, but a gene encoding the TTX-R Na+ channel has not been identified. In this study, we have cloned cDNA encoding the alpha subunit of the TTX-R Na+ channel in NB-1 cells and designated it hNbR1. The longest open reading frame of hNbR1 (accession no. AB158469) encodes 2016 amino acid residues. Sequence analysis has indicated that hNbR1 is highly homologous with human cardiac Nav1.5/SCN5A with > 99% amino acid identity. The presence of a cysteine residue (Cys373) in the pore-loop region of domain I is consistent with the supposition that hNbR1 is resistant to TTX. Analysis of the genomic sequence of SCN5A revealed a new exon encoding S3 and S4 of domain I (exon 6A). In addition, an alternative splicing variant, lacking exon 18, that encodes 54 amino acids in the intracellular loop between domains II and III was found (hNbR1-2; accession no. AB158470). Na+ currents in human embryonic kidney cells (HEK293) transfected with hNbR1 or hNbR1-2 showed electrophysiological properties similar to those for TTX-R I(Na) in NB-1 cells. The IC50 for the TTX block was approximately 8 microM in both variants. These results suggest that SCN5A has a newly identified exon for alternative splicing and is more widely expressed than previously thought.

Amino Acid Sequence↗

Plasmid content and localization of the genes encoding the denitrification enzymes in two strains of Rhodobacter sphaeroides.

Plasmid content and localization of the genes encoding the reductases of the denitrification pathway were determined in the photosynthetic bacterium Rhodobacter sphaeroides forma sp. denitrificans by transverse alternating-field electrophoresis (TAFE) and hybridization with digoxigenin-labeled homologous probes. Two large plasmids of 102 and 115 kb were found. The genes encoding the various reductases are not clustered on a single genetic unit. The nap locus (localized with a napA probe), the nirK gene and the norCB genes encoding the nitrate, nitrite and nitric oxide reductases, respectively, were found on different AseI and SnaBI digested chromosomal DNA fragments, whereas the nos locus (localized with a nosZ probe), encoding the nitrous oxide reductase, was identified on the 115-kb plasmid. Furthermore, the genes encoding two proteins of unknown function, one periplasmic and the other cytoplasmic, but whose synthesis is highly induced by nitrate, were found on a different chromosomal fragment. For comparison, the same experiments were carried out on the well-characterized strain Rhodobacter sphaeroides 2.4.1.

Blotting, Southern↗

Widespread occurrence of phage-encoded exotoxin genes in terrestrial and aquatic environments in Southern California.

Many human diseases are caused by pathogens that produce exotoxins. The genes that encode these exotoxins are frequently encoded by mobile DNA elements such as plasmids or phage. Mobile DNA elements can move exotoxin genes among microbial hosts, converting avirulent bacteria into pathogens. Phage and bacteria from water, soil, and sediment environments represent a potential reservoir of phage- and plasmid-encoded exotoxin genes. The genes encoding exotoxins that are the causes of cholera, diphtheria, enterohemorrhagic diarrhea, and Staphylococcus aureus food poisoning were found in soil, sediment, and water samples by standard PCR assays from locations where the human diseases are uncommon or nonexistent. On average, at least one of the target exotoxin genes was detected in approximately 15% of the more than 300 environmental samples tested. The results of standard PCR assays were confirmed by quantitative PCR (QPCR) and Southern dot blot analyses. Agreement between the results of the standard PCR and QPCR ranged from 63% to 84%; and the agreement between standard PCR and Southern dot blots ranged from 50% to 66%. Both the cholera and shiga exotoxin genes were also found in the free phage DNA fraction. The results indicate that phage-encoded exotoxin genes are widespread and mobile in terrestrial and aquatic environments.

Bacterial Infections↗

Structure and organization of a 25 kbp region of the genome of the photosynthetic green sulfur bacterium Chlorobium vibrioforme containing Mg-chelatase encoding genes.

A region comprising approximately 25 kbp of the genome of the strictly anaerobic and obligate photosynthetic green sulfur bacterium Chlorobium vibrioforme has been mapped, subcloned and partly sequenced. Approximately 15 kbp have been sequenced in it's entirety and three genes with significant homology and feature similarity to the bchI, -D and -H genes and the chlI, -D and -H genes of Rhodobacter and Synechocystis strain PCC6803, respectively, which encode magnesium chelatase subunits, have been identified. Magnesium chelatase catalyzes the insertion of Mg2+ into protoporphyrin IX, and is the first enzyme unique to the (bacterio)chlorophyll specific branch of the porphyrin biosynthetic pathway. The organization of the three Mg-chelatase encoding genes is unique to Chlorobium and suggests that the magnesium chelatase of C. vibrioforme is encoded by a single operon. The analyzed 25 kbp region contains five additional open reading frames, two of which display significant homology and feature similarity to genes encoding lipoamide dehydrogenase and genes with function in purine synthesis, and another three display significant homology to open reading frames with unknown function in distantly related bacteria. Putative E. coli sigma 70-like promoter sequences, ribosome binding sequences and rho-independent transcriptional stop signals within the sequenced 15 kbp region are related to the identified genes and orfs. Southern analysis, restriction mapping and partial sequencing of the remaining ca. 10 kbp of the analyzed 25 kbp region have shown that this part includes the hemA, -C, -D and -B genes (MOBERG and AVISSAR 1994), which encode enzymes with function in the early part of the biosynthetic pathway of porphyrins.

Amino Acid Sequence↗

A mixed-encoding genetic algorithm with beam constraint for conformal radiotherapy treatment planning.

In this paper we propose a new hierarchical evolutionary algorithm that combines binary encoding and floating-point encoding to automatically select the beam directions and determine the weights of the selected beams. With traditional optimization methods the beam directions are fixed a priori by the operator in recognition of the fact that computer selection of beam directions is a difficult problem. In this investigation, we used a hybrid-encoding scheme. The binary encoding part of each chromosome was used to select the beam directions, and its corresponding floating-point encoding part of the same chromosome was used to determine the weights of those selected beams. Before beginning the optimization process, we set a constraint on the number of the beam directions we wanted in the final solution. We present three examples to verify this method. These examples differ with each other in tumor sites, problem sizes, and optimization parameters. Three-dimensional optimization results and statistical data showed that this method is feasible. We think this method can be easily extended to solve more complex target problems (such as nonconvex target problems).

Adolescent↗

Click train encoding in primary auditory cortex of the awake monkey: evidence for two mechanisms subserving pitch perception.

Multiunit activity (MUA) and current source density (CSD) patterns evoked by click trains are examined in primary auditory cortex (A1) of three awake monkeys. Temporal and spectral features of click trains are differentially encoded in A1. Encoding of temporal features occurs at rates of 100-200 Hz through phase-locked activity in the MUA and CSD, is independent of pulse polarity pattern, and occurs in high best frequency (BF) regions of A1. The upper limit of ensemble-wide phase-locking is about 400 Hz in the input to A1, as manifested in the cortical middle laminae CSD and MUA of thalamocortical fibers. In contrast, encoding of spectral features occurs in low BF regions, and resolves both the f0 and harmonics of the stimuli through local maxima of activity determined by the tonotopic organization of the recording sites. High-pass filtered click trains decrease spectral encoding in low BF regions without modifying phase-locked responses in high BF regions. These physiological responses parallel features of human pitch perception for click trains, and support the existence of two distinct physiological mechanisms involved in pitch perception: the first using resolved harmonic components and the second utilizing unresolved harmonics that is based on encoding stimulus waveform periodicity.

Animals↗

A novel thyroid hormone receptor encoded by a cDNA clone from a human testis library.

The c-erbA gene belongs to a multigene family that encodes transcriptional regulatory proteins including the v-erbA oncogene product, steroid hormone receptors, and the vitamin D3 receptor. A v-erbA DNA probe encoding the DNA-binding region of the v-erbA protein was used to screen a human complementary DNA testis library. One of the clones isolated, erbA-T-1, was found to encode a 490-amino acid protein (erbA-T). The erbA-T polypeptide shows high homology with the proteins encoded by both the chicken c-erbA and the human c-erbA-beta genes but is most closely related to the chicken gene. The chicken c-erbA and the human c-erbA-beta genes encode high-affinity receptors for thyroid hormone, and here it is shown that the erbA-T protein binds specifically to 3,5,3'-triiodo-L-thyronine with a dissociation constant of 3.8 +/- 0.2 x 10(-10) M. These data imply that more than one thyroid hormone receptor exists in humans and that these receptors might have different tissue- and gene-activating specificities.

Amino Acid Sequence↗

Age-related reductions in human recognition memory due to impaired encoding.

The participation of the medial temporal cortex and other cerebral structures in the memory impairment that accompanies aging was examined by means of positron emission tomography. Cerebral blood flow (rCBF) was measured during encoding and recognition of faces. Young people showed increased rCBF in the right hippocampus and the left prefrontal and temporal cortices during encoding and in the right prefrontal and parietal cortex during recognition. Old people showed no significant activation in areas activated during encoding in young people but did show right prefrontal activation during recognition. Age-related impairments of memory may be due to a failure to encode the stimuli adequately, which is reflected in the lack of cortical and hippocampal activation during encoding.

Adult↗

Nucleotide sequence and phylogeny of the tet(L) tetracycline resistance determinant encoded by plasmid pSTE1 from Staphylococcus hyicus.

The nucleotide sequence of the tetracycline resistance (tet) gene and its regulatory region, encoded by the plasmid pSTE1 from Staphylococcus hyicus, was determined. The tet gene was inducible by tetracycline and encoded a hydrophobic protein of 458 amino acids. Comparisons between the predicted amino acid sequences of the pSTE1-encoded Tet from S. hyicus and the previously sequenced Tet K variants from Staphylococcus aureus, Tet L variants from Bacillus cereus, Bacillus stearothermophilus, and Bacillus subtilis, Tet M variants from Streptococcus faecalis and Staphylococcus aureus as well as Tet O from Streptococcus mutans were performed. An alignment of Tet amino acid sequences revealed the presence of 30 conserved amino acids among these Tet variants. On the basis of the alignment, a phylogenetic tree was constructed. It demonstrated large evolutionary distances between the Tet M and Tet O variants on one hand and the Tet K and Tet L variants on the other hand. The pSTE1-encoded Tet proved to be closely related to the Tet L proteins originally found on small Bacillus plasmids. The observed extensive similarities in the nucleotide sequences of the tet genes and in the deduced Tet amino acid sequences allowed the assignment of the pSTE1-encoded Tet to the Tet proteins of class L.

Amino Acid Sequence↗

Characterization of a staphylococcal plasmid related to pUB110 and carrying two novel genes, vatC and vgbB, encoding resistance to streptogramins A and B and similar antibiotics.

We isolated and sequenced a plasmid, named pIP1714 (4,978 bp), which specifies resistance to streptogramins A and B and the mixture of these compounds. pIP1714 was isolated from a Staphylococcus cohnii subsp. cohnii strain found in the environment of a hospital where pristinamycin was extensively used. Resistance to both compounds and related antibiotics is encoded by two novel, probably cotranscribed genes, (i) vatC, encoding a 212-amino-acid (aa) acetyltransferase that inactivates streptogramin A and that exhibits 58.2 to 69.8% aa identity with the Vat, VatB, and SatA proteins, and (ii) vgbB, encoding a 295-aa lactonase that inactivates streptogramin B and that shows 67% aa identity with the Vgb lactonase. pIP1714 includes a 2,985-bp fragment also found in two rolling-circle replication and mobilizable plasmids, pUB110 and pBC16, from gram-positive bacteria. In all three plasmids, the common fragment was delimited by two direct repeats of four nucleotides (GGGC) and included (i) putative genes closely related to repB, which encodes a replication protein, and to pre(mob), which encodes a protein required for conjugative mobilization and site-specific recombination, and (ii) sequences very similar to the double- and single-strand origins (dso, ssoU) and the recombination site, RSA. The antibiotic resistance genes repB and pre(mob) carried by each of these plasmids were found in the same transcriptional orientation.

Acetyltransferases↗

Expression of the immunity protein of plantaricin 423, produced by Lactobacillus plantarum 423, and analysis of the plasmid encoding the bacteriocin.

Plantaricin 423 is a class IIa bacteriocin produced by Lactobacillus plantarum isolated from sorghum beer. It has been previously determined that plantaricin 423 is encoded by a plasmid designated pPLA4, which is now completely sequenced. The plantaricin 423 operon shares high sequence similarity with the operons of coagulin, pediocin PA-1, and pediocin AcH, with small differences in the DNA sequence encoding the mature bacteriocin peptide and the immunity protein. Apart from the bacteriocin operon, no significant sequence similarity could be detected between the DNA or translated sequence of pPLA4 and the available DNA or translated sequences of the plasmids encoding pediocin AcH, pediocin PA-1, and coagulin, possibly indicating a different origin. In addition to the bacteriocin operon, sequence analysis of pPLA4 revealed the presence of two open reading frames (ORFs). ORF1 encodes a putative mobilization (Mob) protein that is homologous to the pMV158 superfamily of mobilization proteins. Highest sequence similarity occurred between this protein and the Mob protein of L. plantarum NCDO 1088. ORF2 encodes a putative replication protein that revealed low sequence similarity to replication proteins of plasmids pLME300 from Lactobacillus fermentum and pYIT356 from Lactobacillus casei. The immunity protein of plantaricin 423 contains 109 amino acids. Although plantaricin 423 shares high sequence similarity with the pediocin PA-1 operon, no cross-reactivity was recorded between the immunity proteins of plantaricin 423 and pediocin PA-1.

Amino Acid Sequence↗

Multiple genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase in the gram-positive polychlorinated biphenyl-degrading bacterium Rhodococcus erythropolis TA421, isolated from a termite ecosystem.

Rhodococcus erythropolis TA421 was isolated from a termite ecosystem and is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. Genetic and biochemical analyses of the PCB catabolic pathway of this organism revealed that there are four different bphC genes (bphC1, bphC2, bphC3, and bphC4) which encode 2,3-dihydroxybiphenyl dioxygenases. As determined by Southern hybridization, none of the bphC genes exhibits homology to any other bphC gene. bphC1, bphC2, and bphC4 encode enzymes that have narrow substrate specificities and cleave the first aromatic ring in the meta position. In contrast, bphC3 encodes a meta cleavage dioxygenase with broad substrate specificity. Asturias et al. have shown that the closely related organism Rhodococcus globerulus P6 contains three different bphC genes (bphC1, bphC2, and bpHC3) which encode meta cleavage dioxygenases. The data suggest that there is a diverse family of bphC genes which encode PCB meta cleavage dioxygenases in members of the genus Rhodococcus.

Animals↗

The aflatoxin biosynthesis cluster gene, aflX, encodes an oxidoreductase involved in conversion of versicolorin A to demethylsterigmatocystin.

Biosynthesis of the toxic and carcinogenic aflatoxins by the fungus Aspergillus flavus is a complicated process involving more that 27 enzymes and regulatory factors encoded by a clustered group of genes. Previous studies found that three enzymes, encoded by verA, ver-1, and aflY, are required for conversion of versicolorin A (VA), to demethylsterigmatocystin. We now show that a fourth enzyme, encoded by the previously uncharacterized gene, aflX (ordB), is also required for this conversion. A homolog of this gene, stcQ, is present in the A. nidulans sterigmatocystin (ST) biosynthesis cluster. Disruption of aflX in Aspergillus flavus gave transformants that accumulated approximately 4-fold more VA and fourfold less aflatoxin than the untransformed strain. Southern and Northern blot analyses confirmed that aflX was the only gene disrupted in these transformants. Feeding ST or O-methylsterigmatocystin, but not VA or earlier precursor metabolites, restored normal levels of AF production. The protein encoded by aflX is predicted to have domains typical of an NADH-dependent oxidoreductase. It has 27% amino acid identity to a protein encoded by the aflatoxin cluster gene, aflO (avfA). Some of domains in the protein are similar to those of epoxide hydrolases.

Amino Acid Sequence↗

D-xylose metabolism in Hypocrea jecorina: loss of the xylitol dehydrogenase step can be partially compensated for by lad1-encoded L-arabinitol-4-dehydrogenase.

With the goal of the genetic characterization of the D-xylose pathway in Hypocrea jecorina (anamorph: Trichoderma reesei), we cloned the xdh1 gene, encoding NAD-xylitol dehydrogenase, which catalyzes the second step of fungal D-xylose catabolism. This gene encodes a 363-amino-acid protein which has a mass of 38 kDa, belongs to the zinc-containing alcohol dehydrogenase family, exhibits high sequence identity to the published sequences of xylitol dehydrogenases from yeast origins, but contains a second, additional binding site for Zn2+. The enzyme catalyzed the NAD-dependent oxidation of xylitol and D-sorbitol and the NADH-dependent reduction of D-xylulose and D-fructose. No activity was observed with NADP, L-arabinose, or L-arabinitol. A single 1.4-kb transcript was formed during growth on xylan, D-xylose, L-arabinose, L-arabinitol and, at a lower abundance, xylitol, D-galactose, galactitol, and lactose but not on D-glucose and glycerol. xdh1 deletion mutants exhibited 50% reduced growth rates on D-xylose, whereas growth rates on xylitol remained unaltered. These mutants contained 30% of the xylitol dehydrogenase activity of the parent strain, indicating the presence of a second xylitol dehydrogenase. This activity was shown to be due to lad1-encoded L-arabinitol-4-dehydrogenase, because H. jecorina xdh1 lad1 double-deletion strains failed to grow on D-xylose or xylitol. In contrast, lad1 deletion strains of H. jecorina grew normally on these carbon sources. These results show that H. jecorina contains a single xylitol dehydrogenase which is encoded by xdh1 and is involved in the metabolism of D-xylose and that lad1-encoded L-arabinitol-4-dehydrogenase can compensate for it partially in mutants with a loss of xdh1 function.

Amino Acid Sequence↗

Distribution and degree of heterogeneity of the afimbrial-adhesin-encoding operon (afa) among uropathogenic Escherichia coli isolates.

The afimbrial adhesin (AFA-I) from a pyelonephritic Escherichia coli isolate (KS52) is a mannose-resistant, P-independent, X-binding adhesin, expressed by the afa-1 operon. It is distinct from the E. coli X-binding adhesins with M and S specificity. A total of 138 E. coli isolates belonging to various serotypes, mostly from urinary tract infections, were screened for the presence of DNA sequences related to the afa operon and for the expression of an X-adhesin able to mediate mannose-resistant hemagglutination (MRHA) and adhesion to uroepithelial cells. Fifteen strains were shown to harbor DNA sequences related to the AFA-I-encoding operon, and 13 of them expressed an X-adhesin. Using as probes different DNA segments of the AFA-I-encoding operon in Southern experiments, we demonstrated that only three of these clinical isolates contained genetic determinants closely related to those identified in the original afa prototype strain (KS52): presence of the afaA, afaB, afaC, afaD, and afaE genes associated with the expression of a 16,000-dalton hemagglutinin-adhesin which strongly cross-reacted with AFA-I-specific antibodies. The other E. coli isolates harbored DNA sequences homologous to the afaA, afaB, afaC, and afaD genes, but lacked the sequence corresponding to the adhesin-producing gene afaE; Western blots allowed the detection of polypeptides (15,000, 15,500, or 16,000 daltons) in these strains which cross-reacted with variable intensity with antibodies raised against the denatured AFA-I protein, but did not cross-react with native AFA-I-specific antibodies. Following DNA cloning experiments from chromosomal DNA of two of those strains (A22 and A30), we demonstrated that although the AFA-related operon in A22 and A30 strains lacked the AFA-I adhesin-encoding gene, they synthesized a functional X-adhesin. Thus, strains A22 and A30 encode adhesins designated AFA-II and AFA-III, which were cloned on recombinant plasmids pILL72 and pILL61, respectively. Southern hybridization experiments and Western blot analyses of the 15 AFA-related strains demonstrate the heterogeneity of the genetic sequences encoding the structural adhesin and suggest the bases for the serological diversity of the AFA adhesins.

Adhesins, Escherichia coli↗

Cloning and nucleotide sequence of the gene (trh) encoding the hemolysin related to the thermostable direct hemolysin of Vibrio parahaemolyticus.

Vibrio parahaemolyticus isolates derived from an outbreak of gastroenteritis in the Republic of Maldives did not have the genetic potential to produce the thermostable direct hemolysin, but one such isolate produced a hemolysin immunologically related to the thermostable direct hemolysin (T. Honda, Y. Ni, and T. Miwatani, Infect. Immun. 56:61-965, 1988). The Maldives isolates hybridized with the DNA probe for the gene encoding the thermostable direct hemolysin (the tdh gene) under reduced stringencies. A DNA fragment containing the probe-reactive nucleotide sequence was isolated from a selected strain and cloned into pBR322 in Escherichia coli. A clone producing the thermostable direct hemolysin-related hemolysin was obtained by screening with hemolysis assays and by an immunological assay. Nucleotide sequence analysis of the cloned DNA fragment revealed that the gene encoding the thermostable direct hemolysin-related hemolysin (the trh gene), like the tdh gene, encoded the hemolysin subunit composed of 189 amino acid residues. The trh gene had significant nucleotide sequence homology with the tdh gene (68.4% with the tdh1 gene copy and 68.6% with the tdh2 gene copy). The amino acid sequences of the hemolysin subunits deduced from the nucleotide sequences of the trh gene and tdh gene were homologous (61.9% homology with the tdh1-encoded subunit and 63.0% homology with the tdh2-encoded subunit) and contained the two cysteine residues to form an intrachain bond at the same positions, and their possible conformations appeared to be similar as determined by hydrophobicity-hydrophilicity analysis and a secondary structure prediction. The trh and tdh genes may have had a common ancestor and may have evolved by single-base changes so that they maintained the fundamental architecture of the molecules.

Amino Acid Sequence↗

Genetic and DNA sequence analysis of the Salmonella typhimurium virulence plasmid gene encoding the 28,000-molecular-weight protein.

We have confirmed that the 28,000-molecular-weight (28K) protein encoded by the virA gene of the 90-kilobase Salmonella typhimurium virulence plasmid is a virulence factor. It was previously shown that a Tn5 insertion, vir-22::Tn5, located in the virulence plasmid greatly attenuated virulence for mice and inhibited the production of a 28K protein (P.A. Gulig and R. Curtiss III, Infect. Immun. 56:3262-3271, 1988). Plasmid pYA426 fully complemented vir-22::Tn5 to virulence by increasing splenic infection after oral inoculation and encoded the 28K protein. To identify the virulence gene(s) of pYA426 mutated by vir-22::Tn5, we constructed nested deletions in pYA426 and examined deletion derivatives for their abilities to complement vir-22::Tn5. Only derivatives still producing the 28K protein complemented vir-22::Tn5. Furthermore, the smallest complementing derivative encoded only the 28K protein, as determined by DNA sequence analysis. Therefore, the 28K protein is sufficient for complementation of the attenuating mutation vir-22::Tn5 and must be the virulence factor inhibited by the insertion. We determined the nucleotide sequence of the 1.2-kilobase BamHI-EcoRI fragment encoding the 28K protein and identified the structural gene, virA. A 723-base-pair open reading frame which encodes a peptide with a molecular weight of 27,572 was found.

Amino Acid Sequence↗