Analysis of class I introns in a mitochondrial plasmid associated with senescence of Podospora anserina reveals extraordinary resemblance to the Tetrahymena ribosomal intron

Summary Recently, the nucleotide sequences for three mitochondrial plasmids associated with senescence of Podospora anserina were determined (Cummings et al. 1985). One of these sequences, corresponding to the plasmid termed senDNA, contains three class I introns, all within a protein coding sequence equivalent to the mammalian URF1 gene. Here, we present primary and secondary structure analyses for two of these introns as well as a partial analysis for the third, which extends beyond the DNA sequence determined. With regard to both primary and secondary structure, the closest known relative of intron 1 is the self-splicing intron in the large ribosomal RNA gene of Tetrahymena. One secondary structure domain at the periphery of intron 1 and Tetrahymena models is also present in intron 2. The latter intron is the longest known class I member and contains remnants of two protein-coding sequences, one of which is split by the other. Evolutionary processes that might be responsible for the unusual structure of introns 1 and 2 are discussed.     

Molecular and cytogenetic organization of the 5S ribosomal DNA array in chicken (Gallus gallus)

The 5S ribosomal (r) RNA genes encode a small (120-bp) highly-conserved component of the large ribosomal subunit. The objective of the present research was to study the molecular and cytogenetic organization of the chicken 5S rDNA. A predominant 2.2-kb gene (5S) consisting of a coding and intergenic spacer (IGS) region was identified in ten research and commercial populations. A variant gene repeat of 0.6kb (5S) was observed in some of the populations. Genetic linkage analysis and cytogenetic localization by fluorescence in-situ hybridization assigned the 5S rDNA to chromosome 9. The 5S rDNA array was determined to be 80.2±7.0kb upon electrophoretic sizing following EcoRV digestion. Sequence analysis of 5S IGS regions revealed considerable conservation between chicken subspecies (98.4% identity) as well as homology with vertebrate Pol III promoter and regulatory sequence motifs. Minor intraindividual sequence variation within 1000bp of IGS was observed in four cloned Red Jungle Fowl (Gallus gallus gallus) 5S repeats (95.5% identity in this region). Sequence comparisons between IGS regions of 5S and 5S genes indicated two short continuous (>20bp) and many short non-continuous homologous regions as well as other conserved features such as promoter and termination motifs.     

Reiterated sequences of herpes simplex virus type 1 (HSV-1) genome can serve as physical markers for the differentiation of HSV-1 strains

Summary The stability of regions containing tandemly reiterated sequences in the S component of the herpes simplex virus type 1 (HSV-1) genome was determined, by comparing restriction fragments of the regions among sets of HSV-1 isolates derived from a single source. The 6 reiterations examined were grouped into three. Reiteration VII (within protein coding regions of genes US10 and US11) and reiteration IV (within introns of genes US1 and US12) were stable between the isolates (group 1). Regions containing one of four other reiterations were detected as a set of ladder-like fragments. Reiteration II (between a sequence and IE175 gene) and reiteration VI (within an intergenic region on the 3 side of the 3 co-terminal family of genes US10, US11, and US12) (group 3) were more unstable than reiteration I (within a sequence) and reiteration III (between a sequence and IE175 gene) (group 2). The mode of fluctuation of the reiterations observed within a set of HSV-1 strains isolated from an individual was similar to that observed between HSV-1 single-plaque clones separated in cultured cells. These reiterations, except for group 3, can serve as sensitive and convenient markers for differentiating HSV-1 strains.     

Group-I intron family in the nuclear ribosomal RNA small subunit genes ofCenococcum geophilum isolates

A family of optional group-I introns was found near the 3 end of the nuclear small subunit rRNA genes in 61 out of 70 isolates of the deuteromycete mycorrhizal fungusCenococcum geophilum. DNA sequence polymorphisms among the introns (termedCgSSU introns) from ten of the isolates were studied. The sequences, ranging in size from 488 to 514 nucleotides, were from 93.2% to 99.6% similar to each other. Mutations were less common in predicted base-paired regions (33% of all mutations) than in free-standing regions (67%). The introns were self-spliced in vitro and were closest to subgroup ICI according to sequence and predicted secondary structure. Group-I intron pairing regions P1 through P10, including core regions P, Q, R and S, were present in all tenCgSSU introns studied. No lengthy open reading frames were found in any of the introns, indicating that the introns do not encode a protein, and therefore may not be mobile. It is likely that a single intron entered a progenote ofC. geophilum and changed as the species evolved.     

Heterogeneity of intron presence or absence in rDNA genes of the lichen species Physcia aipolia and P. stellaris

Intron origin and evolution are of high interest, yet the rates of insertion and loss are unclear. To investigate their spread, we studied ribosomal (r)DNA introns from the closely related lichens Physcia aipolia and P. stellaris. Both taxa are replete with rDNA spliceosomal introns and autocatalytic group I introns, many of which show presence/absence polymorphism when screened with the PCR approach. This initially suggested that Physcia could be a model for studying intron retention and loss. However, during the course of a population-level analysis, we discovered widespread intron presence/absence heterogeneity within lichen thalli. To address this result, we sequenced multiple clones encoding nuclear rDNA and the single-copy elongation factor-1 (EF-1) from individual thalli. These data showed extensive rDNA heterogeneity within individuals, rather than the presence of multiple fungi within a thallus. Our results suggest that considerable care must be taken when interpreting intron presence/absence in lichen rDNA, an observation that has general implications for the study of rDNA intron evolution.     

Discrete functional contributions of cerebral cortical foci in voluntary swallowing: a functional magnetic resonance imaging (fMRI) “Go,No-Go” study

Brain-imaging studies have shown that visually-cued, voluntary swallowing activates a distributed network of cortical regions including the precentral and postcentral gyri, anterior cingulate cortex (ACC), insula, frontoparietal operculum, cuneus and precuneus. To elucidate the functional contributions of these discrete activation foci for swallowing, a Go, No-Go functional magnetic resonance imaging (fMRI) paradigm was designed. Brain activation associated with visually-cued swallowing was compared with brain activation evoked by a comparable visual cue instructing the subject not to swallow. Region-of-interest analyses performed on data from eight healthy subjects showed a significantly greater number of activated voxels within the precentral gyrus, postcentral gyrus, and ACC during the Go condition compared to the No-Go condition. This finding suggests that the precentral gyrus, postcentral gyrus, and ACC contribute primarily to the act of swallowing. In contrast, the numbers of activated voxels within the cuneus and precuneus were not significantly different for the Go and No-Go conditions, suggesting that these regions mediate processing of the cue to swallow. Together these findings support the view that the discrete cortical foci previously implicated in swallowing mediate functionally distinct components of the swallowing act.     

The presequence of cytochrome c1 from potato mitochondria is encoded on four exons

The structural organization of a nuclear gene encoding cytochrome c₁ from potato was determined. The gene spans 5.1 kb and contains eight introns. All intron/exon junctions follow the GT/AG rule. Functional domains of the mature cytochrome c₁ protein are located on separate exons. The presequence, which targets the cytochrome c₁ precursor to the mitochondrion and to the correct intra-mitochondrial location, is encoded on the first four exons. The largest intron (2.8 kb) separates the information for mitochondrial targeting from the intra-mitochondrial sorting domain of the cytochrome c₁ protein. In contrast to other organellar precursor proteins, there is no intron between the DNA sequence encoding the presequence and the mature protein. This may indicate that during evolution the genetic information for the prokaryotic cytochrome c₁ was transferred to the nucleus together with the bacterial secretion signal which is structurally and functionally related to intramitochondrial sorting domains.     

Lability in the responses of cells in the auditory cortex of squirrel monkeys to species-specific vocalizations

Summary The activity of 28 cells located mainly in the secondary auditory cortex (A II) of awake squirrel-monkeys, was extracellularly recorded for periods of up to 6 h. Seven different species-specific vocalizations, which were repeatedly presented to the monkey, were used as auditory stimuli. Twenty-six cells responded, at least once, to one or more vocalizations; 22 cells revealed some change in their response (pattern or strength) to at least one vocalization (change in response). Twenty-one cells exhibited a change in the number and/or type of vocalization to which they responded during the recording period (change in selectivity). At some time during the recording period all the responding cells exhibited a change in response and/or a change in selectivity (change in responsiveness). A change in response of a cell to a vocalization did not necessarily exclude a change in selectivity, associated with the same vocalization, later in time and vice-versa. A change in responsiveness to one vocalization was not necessarily correlated with changes in responsiveness to other vocalizations.     

An improved apparatus for the optical recording of contraction of single heart cells

An optical system for measuring changes in cell length during unloaded contractions of cardiac myocytes is described. A one-dimensional video image of a cell is obtained every 4 ms with a linear photodiode array, which is aligned with the longitudinal axis of the cell. The circuit used to process the image from the photodiode array has a variety of features to aid in the accurate determination of the distance between the ends of the cell, i.e. the cell length. First, the video image of the cell is divided into two windows, one encompassing the front edge of the cell, the other encompassing the rear edge. Other cells or debris beyond the cell edges are excluded. Changes in the general light level, for example as a result of debris floating above the cell, have little effect because within the windows the background light level is subtracted from the signals before they are processed further. To detect the cell edges, the system determines when the signals within the windows exceed (front edge) or drop below (rear edge) chosen threscholds, which are different for the front and rear edges. The system has memory and it identifies the rear edge of the cell as the last time the signal falls below the threshold; because of this bright spots within the cell are not mistaken for the end of the cell. The system has hysteresis, which enables it to ignore small fluctuations in brightness around the threshold. The system is easy to use, accurate, readily calibrated, and it has good spatial and time resolution (about 0.25 m and 4 ms respectively).     

Brief Communication: cDNA Sequence and Mapping of the Mouse Copb Gene Encoding the Beta Subunit of the COPI Coatomer Complex

COPI-coated vesicles are involved in retrograde-directed selective transport of proteins from the Golgi complex to the endoplasmic reticulum (ER) as well as mediate anterograde transport of cargo proteins within the Golgi or in endosomal trafficking. The COPI protein complex contains an ADP-ribosylation factor (ARF1) and seven coatamer subunits (, , , , , , -COP). The localization and function of human subunit of coatamer (COPB) suggests it is likely a candidate gene of ruby-eye-2 (ru2), which is a mouse model of human Hermansky-Pudlak syndrome characterized by the dysfunction of several subcellular organelles. In this study, we determined the entire coding sequence of mouse (Copb) cDNA by combining an overlapping mouse EST contig with EST walking. -COP was found highly conserved in mouse, rat, and human, and it is ubiquitously expressed in mouse. The Copb gene was mapped to mouse Chr 7 at a position of 53.3 cM by radiation hybrid mapping. Our RH mapping data, sequencing of RT-PCR products, and Western blotting exclude the Copb gene as a candidate for ru2.     

Neurons sensitive to narrow ranges of repetitive acoustic transients in the medial geniculate body of the cat

Summary Neuronal activity was recorded in the medial geniculate body (MGB) of nitrous oxide anaesthetized, paralysed cats in response to click trains. For most cells responding to these stimuli the spike discharges are precisely time locked to individual clicks within the train. The present study has revealed that, apart from the normal locker response being characterized by a monotonic decrease in the entrainment as the frequency of the clicks within the train increases, there is a small population of lockers which show a non-monotonic response to increasing click frequency. 41% of these non-monotonic cells were not at all entrained by the lowest click rates and had time-locked responses for very restricted frequency ranges. These particular non-monotonic lockers were more commonly-found in the posterior part of the pars lateralis and in the suprageniculate nucleus. These cells might be involved in the temporal coding of natural sounds such as animal vocalizations and the cat's purr.Supported by the Swiss National Science Foundation, grant no. 3.509.79     

Application of the polymerase chain reaction to study the M protein(-like) gene family in beta-hemolytic streptococci

Evaluation of homologous regions of published M protein (emm) gene sequences from group A streptococci (GAS; Streptococcus pyogenes) was used to design three primer pairs for polymerase chain reaction (PCR) and three oligonucleotide probe sequences internal to the amplified products. One set of primers and corresponding probe should detect and lead to amplification of emm(-like) genes of virtually every type (all M), another (SOR^-M) should only amplify emm(-like) genes from GAS negative for serum opacity reaction (SOR) and the third (SOR⁺M) should expand only emm(-like) genes from SOR⁺ GAS. Using the all M primer pair for PCR on the genomic DNA from GAS of 29 different M types as well as from a group C and a group G streptococcal isolate, DNA fragments within the expected size range were amplified in every assay. All PCR products reacted with the all M probe. Related sequences were not detected in genomic DNA of an S. agalactiae and an Enterococcus faecalis isolate. Applying the SOR^-M and SOR⁺M primers to identical assays led to mutually exclusive amplification products. The SOR⁺M and SOR⁺M probes hybridized only to their corresponding products. Exceptions to this exclusivity were the SOR⁺ GAS of M types 3, 8, 27, 34, 42, 67, and 69, which consistently reacted only with the SOR⁺M primer/probe set. Analysis of sequence data from the amplified emm(-like) 2, 3, 18, and 19 genes revealed interesting specific features such as conserved gaps in the C-terminal sequence regions from SOR⁺ and the exceptional SOR^- GAS strains. These data indicate the existence of a subgroup of strains among SOR^- GAS and may advance our understanding of phylogenetic relationship between different serotypes of GAS.     

Analysis of the introns in genes encoding small G proteins

Because all small G proteins (SGPs) possess a very similar array of structural and functional domains, they are obvious candidates for examining the relationships postulated to exist between the exon-intron structure of genes and the domain structure of the encoded proteins. To address this issue, and to possibly gain insight into the evolution of their introns, we have analyzed positions, sizes, and sequences of 125 introns from 28 SGP genes. These introns were found to be distributed in 60 different locations throughout the aligned sequences, with a preference for the 5-half of the genes. More than 50% of the positions were found to be shared by two or more genes, and genes encoding SGPs of very similar amino acid sequence (i.e., isotypes) in quite closely related species tend to have most, or all, of their introns in identical locations, indicating a common evolutionary origin (homologous introns). However, with few exceptions, no statistically significant sequence similarity or common folding motif was found between homologous intron pairs. Only three intron positions are shared between members of distantly related SGP subfamilies. These three potentially ancient intron locations fall between regions encoding -helices or -sheets, but two of them interrupt regions encoding known functional (guanosine-nucleotide-binding) modules. Intron positions that are occupied only in single genes, or in genes encoding very similar SGPs, do not show any preferential distribution with respect to regions encoding structural or functional motifs. This discordance between exon modules and structural and/or functional protein domains suggests that most, if not all, introns in modern SGP genes arose by independent insertion events after diversification of the various SGP subfamilies, and therefore probably did not participate in the early evolution of these genes.     

The complete nucleotide sequence of the genome RNA of Lily symptomless virus and its comparison with that of other carlaviruses

Summary. The complete genomic nucleotide sequence and genome structure of Lily symptomless virus (LSV), a lily-infecting carlavirus, have been obtained. The genome of the Korean strain of LSV, LSV-Kr, was 8,394 nucleotides long and contained six open reading frames (ORFs) coding for proteins of Mr 220kDa (1,948aa), 25kDa (228aa), 12kDa (106aa), 7kDa (64aa), 32kDa (291aa) and 16kDa (140aa) from the 5 to 3 end, respectively, which is typical of carlaviruses. Genetic heterogeneity was observed in the ORF1 gene. A total of 221 of 5,847 nucleotides (nt) were heterologous in the ORF1 of replicase; 162nt portions were silent and 59nt resulted in amino acid changes. This heterogeneity indicates that the LSV-infecting lily plants contained a genetically heterogeneous population of LSV (quasispecies). Overall similarities to those of other carlaviruses for the six ORFs of LSV were from 76.1% to 31.6% and from 87.3% to 13.7%, at nucleotide and amino acid levels, respectively. The ORF1 replicase gene of LSV shares 40.9% to 56.8% and 48.9% and 58.6% identities with that of 5 other carlaviruses at the amino acid and nucleotide levels, respectively. LSV was closest to Blueberry scorch virus (BlScV) in this ORF, among the carlaviruses for which sequence information is available. The three triple gene blocks (ORF2-4), ORF5 (coat protein) and 3-proximal 16kDa ORF6 genes were further analyzed, and phylogenetic trees for the coding regions indicate that the LSV was the most closely related to Kalanchoe latent virus and BlScV. This is the first report of the complete nucleotide sequence and genome structure of LSV.Received December 13, 2002; accepted May 14, 2003 Published online July 17, 2003     

Firing characteristics of vestibular nuclei neurons in the alert monkey after bilateral vestibular neurectomy

Summary After destruction of the peripheral vestibular system which is not activated by moving large-field visual stimulation, not only labyrinthine-ocular reflexes but also optokinetic-ocular responses related to the velocity storage mechanism are abolished. In the normal monkey optokinetic-ocular responses are reflected in sustained activity changes of central vestibular neurons within the vestibular nuclei. To account for the loss of optokinetic responses after labyrinthectomy, inactivation of central vestibular neurons consequent on the loss of primary vestibular activity is assumed to be of major importance. To test this hypothesis we recorded the neural activity within the vestibular nuclear complex in two chronically prepared Rhesus monkeys during a period from one up to 9 and 12 months after both vestibular nerves had been cut. The discharge characteristics of 829 cells were studied in relation to eye fixation, and to a moving small and large (optokinetic) visual stimulus producing smooth pursuit (SP) eye movements and optokinetic nystagmus (OKN). Units were grouped into different subclasses.After chronic bilateral vestibular neurectomy (BVN) we have found: (1) a rich variety of spontaneously active cells within the vestibular nuclear complex, which — as far as comparison before and after BVN is possible — belong to all subclasses of neurons functionally defined in normal monkey; and (2) no sustained activity changes which are related to the activation of the velocity storage mechanism; this is especially true for pure-vestibular, vestibular-pause and tonic-vestibular-pause cells in normal monkey which show a pure, pause and tonic-pause firing pattern after BVN. Neurons which are modulated by eye position are, however, modulated with the velocity of slow eye movements with comparable sensitivity during SP and OKN. Retinal slip is extremely rarely encoded. The results of the present study do not directly answer the question why the velocity storage mechanism is abolished after BVN but they suggest that only a small number of central vestibular cells may be inactivated by neurectomy.Supported by SNF grant no. 3.510-0.86     

Effects of sleep deprivation on the activity of selected metabolic enzymes in skeletal muscle

Summary The present study gives the results of a comparison of the recorded and true tibia-calcaneal angles in 17 normal subjects and in 14 patients with abnormally hypoextensible non contracting triceps. 1. For a minimal passive torque, the difference between true and recorded angles varied considerably from one individual to another. The means and ranges for the two groups were respectively: –8 (+7, –21) and –7 (+5, –20). 2. When the passive torque increased as a result of slow passive lengthening of the muscle, the true curve was steeper than the recorded one, owing to differences between the two angle measurements. For each of the two groups the differences in means and ranges were respectively: 6 (0, +13.5) and 8 (3, 12). 3. Subjects made isometric voluntary contractions of the triceps surae at fixed angles which corresponded to step by step muscle lengthening. The resulting true curve was much steeper than the recorded curve. The differences in means and ranges were: 7 (1.5, +15) in children of the two groups and respectively 3 (0, +9) and 12 (10, 14) in adults of the two groups. The present results show that this methodology was the only reliable way of correctly obtaining passive and active torque-angle curves, measuring differences between subjects, appreciating the effects of treatments and these by ascertaining whether or not trophic muscle regulation was defective.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Acetylcholine metabolism in the brain of rats treated with vanadyl sulfate and certain phenylacetic acid derivatives

Summary The present work describes the effect produced by phenylacetic acid, phenylethylacetic and diphenylacetic acids (sodium salts) separated and together with vanadyl sulfate on some indices, of acetylcholine metabolism in the rat's brain. Total cholinesterase activity and the free and bound (conditionally) acetylcholine levels served as indices. As shown experimentally the use of phenylacetic acid derivatives is accompanied by reduction of the bound acetylcholine, content, whereas that of free acetylcholine and the total tissue cholinesterase activity remain unchanged. Vanadyl sulfate provokes a significant reduction of the total tissue cholinesterasic activity, but does not change the content of free and bound acetylcholine therein. In conjoint action of the above-mentioned substances, the effects of phenylacetic acid erivatives are supplemented by the anticholinesterase effect of vanadyl sulfate.(Presented by Active Member AMN SSSR S. V. Anichkov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 57, No. 2, pp. 80–83, February, 1964     

Neuron Activity in the Prefrontal Cortex of the Brain in Rats with Different Typological Characteristics in Conditions of Emotional Stimulation

Male Wistar rats were separated according to the emotional resonance method (groups of animals avoiding (altruists) and not avoiding (egotists) the pain cries of partner rats) and neuron activity in the prefrontal areas of the cortex was studied in the right and left hemispheres. Assessments were made of changes in the frequency of nerve cell spike activity (in relation to the baseline activity of neurons in sated animals) in rats subjected to one day of food deprivation and after electrical stimulation of emotionally positive (lateral hypothalamus) and negative (tegmentum of the midbrain) brain structures and after exposure to the pain cries of partner rats. The results of these experiments revealed a series of differences in the cell activities of the two groups of rats. In conditions of hunger, the discharge frequency in the altruists was higher than that in egotists. Cortical neuron responses to positive stimulation were greater than those to negative stimulation in rats of both groups. Intracerebral stimulation produced significantly greater increases in discharge frequency in neurons of both prefrontal areas of the cortex in altruists than in egotists. In both groups of rats, neurons in the right hemisphere responded to emotionally negative stimulation with significantly greater activation than cells in the left hemisphere, while activity in the left hemisphere was greater in conditions of emotionally positive stimulation. Altruists showed significantly greater neuron responses during exposure to pain cries from victim rats in both the right and left hemispheres. The responses of egotists to victim cries were not significantly different from baseline activity levels.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.