Repetitive DNA insertion in a protein kinase ORF of a latent FSV (Feldmannia sp. virus) genome.

We describe the structure of a Feldmannia sp. virus (FsV) genome integrated in the brown alga, Feldmannia. This integrated FsV genome appears to be permanently inactivated and lost its ability to excise and replicate. Unlike the replicated form of FsV, this integrated FsV genome contains a large (>50 kb) repeat region inserted in a protein kinase open reading frame. While related to the 173-bp repeats previously characterized in the FsV genome (Lee et al., 1995), Southern blot analysis indicates that the repeats in the inactive, integrated FsV genome are distinct from those previously characterized. Fine structural analysis of the repeat-insertion sites in the protein kinase gene indicates that there are 8- and 10-bp palindromic sequences present in multiple locations located near the repeat-insertion site. The translated protein kinase contains all of the catalytic motifs conserved in most serine/threonine protein kinases and a potential autophosphorylation site. This protein kinase gene is expressed as RNA in sporophyte plants where virus production is active but not in gametophyte plants where the virus genome is latent. The structure of the integrated virus genome is discussed.

A Feldmannia algal virus has two genome size-classes.

Persistent viruses occur intracellularly in brown algae, specifically the Ectocarpales, and as reported here in the genus Feldmannia. Feldmannia species are small (1 mm-several cm), filamentous forms with single-celled meiotic sporangia that normally produce haploid zoospores. In the isolate reported here, spores were not observed in the sporangia but rather numerous (approximately 10(6) per cell) polyhedral viruses are formed in their place. Two dsDNA genome classes of 158 and 178 kbp, with two restriction site variants of each, are described. The individual abundance of each genome in viral preparations is affected by culture temperature. A cosmid library was used to generate circular restriction enzyme (BamHi, Noti, and Psti) site maps.

A brown algal virus genome contains a "RING" zinc finger motif.

The brown filamentous alga Feldmannia sp. contains a large icosahedral dsDNA virus, FsV, of which there are multiple variants. A 4.5-kb SstI-HindIII fragment (SH4.5) that is conserved among all genome variants was sequenced. Three open reading frames (ORF-1, -2, and -3, containing 555, 2022, and 411 bp, respectively) were shown to be transcriptionally active by ribonuclease protection assay. A "RING" zinc finger motif and a nucleotide binding site motif were identified in ORF-2.

Characterization of a repetitive DNA element in a brown algal virus.

We describe a family of repetitive sequences found in viruses infecting the brown alga Feldmannia sp. Previously we have demonstrated that the dsDNA genomes of viruses infecting one Feldmannia sp. isolate exist as two size classes of 160 and 179 kb. Repetitive sequences within these genomes were first demonstrated based on the anomalous hybridization among five BamHI fragments from digested virus DNA. Sequence analysis of one of those fragments, B2.4, revealed the presence of 173-bp direct repeats. The restriction maps of the cross-hybridizing BamHI fragments in the two FsV (Feldmannia sp. virus) genome size classes show that these repeats are not widely dispersed, rather they are confined to a small region of each virus genome. We estimate the number of these repeats in the 179-kb genome to be about 109 and in the 169-kb genome to be about 41. In the 179-kb genome, the repeats are contained within a 22-kb region, about 12% of that virus genome, and in the 160-kb genome the repeats are contained within a 10-kb region, about 6% of the genome. The difference in repeat numbers can account for 62% of the size difference between the two size classes of the FsV genome.

Cloning of a chromosomal gene required for phage infection of Lactococcus lactis subsp. lactis C2.

A phage-resistant mutant with a defect in a membrane component required for phage infections in Lactococcus lactis subsp. lactis C2 was transformed with a chromosomal library of the wild-type, phage-sensitive strain. Of the 4,200 transformants screened for phage sensitivity, three were positively identified as phage sensitive. A cause-and-effect relationship between the cloned chromosomal fragments and the phage-sensitive phenotype was established on the basis of the following two criteria: (i) the frequency of loss of the cloned fragments in the absence of antibiotic selection pressure correlated with the frequency of loss of phage sensitivity; and (ii) phage sensitivity was transferred to 100% of recipient, phage-resistant cells transformed with the cloned fragment. The cloned chromosomal DNA from the three independent isolates was physically mapped with restriction endonucleases. The sizes of the cloned fragments were 9.6, 11.8, and 9.5 kb. Each fragment contained an identical stretch of DNA common to all three, which was 9.4 kb. The gene that conferred phage sensitivity was localized by subcloning to a 4.5-kb region. Further subcloning indicated that a single EcoRI site within the 4.5-kb region must lie within the gene or its promoter. The required 4.5-kb region was sequenced and found to code for one partial and two complete open reading frames. The gene required for complementation was functionally mapped by Tn5 mutagenesis and localized to one of the two complete open reading frames, which was designated pip (an acronym for phage infection protein). pip is 2,703 bases in length. Potential promoters start 206 and 212 bases upstream of the open reading frame. A ribosome binding site and a seven-base spacer precede the GTG (Val) translation initiation codon. The amino acid sequence deduced from the gene has 901 residues and an M(r) of 99,426. Hydropathy analysis revealed four to six potential membrane-spanning regions, one near the amino terminus and the others at the extreme carboxyl terminus. The amino terminus has characteristics of a signal sequence. The putative protein would have a 650-residue, central polar domain.

P300 response: habituation.

This study used the "oddball" counting paradigm to examine the possible habituation of the auditory P300 response. Twenty subjects kept a mental record of the number of rarely occurring tone pips presented in a series of more frequently occurring tone pips. Data were collected continuously until responses to 150 rare tone pips were obtained. Findings indicated that the P300 complex decreased in amplitude as a result of repeated stimulation. The decline was logarithmic, not linear, which suggests a stabilization of the amplitude over time. We suggest that the attenuation of amplitude was habituation and not a result of a recovery cycle.

Middle-component AERs during backward masking.

This study examined the middle-component averaged electroencephalic response (AER) to tonal stimuli presented in a backward-masking paradigm. Ss were 4 normal-hearing young adults. Tone-Only (TO), Masker-Only (MO), and Tone-Masker (TM) responses were collected. The visual impression was that the TO response was not significantly different from the extracted response to the tone (T) from the TM response. This result obtained when the tone in the TM condition was presented either slightly above (+5 db) or slightly below (-5 db) perceptual threshold. Backward masking was discussed in terms of two contrasting views of the auditory system: the classical auditory nervous system alone and a parallel system combining the classical auditory and the reticular sensory systems. Speculations were made for each system regarding the type of AER expected for a stimulus submerged in backward masking. The mechanism responsible for backward masking was seen as a preperceptual device that allows discrete samples of the auditory environment to be processed. This mechanism was discussed in terms of its possible relation to forward masking and to loudness enhancement.

The acoustic reflex and temporary threshold shift: temporal characteristics.

One ear of each of seven normal-hearing subjects was exposed to a continuous 1000-Hz tone at 110 dB SPL for three minutes. During exposure, a broad-band noise at 100 dB SPL was presented to the contralateral ear. The noise was either continuous or pulsed. Four pulsed conditions employed repetition periods of 360, 180, 90, or 9 msec with a 50% duty cycle. A control condition in which no noise was presented was also included. Temporary threshold shift was measured at selected postexposure times at the frequency one-half octave above the exposure frequency. TTS2 was greatest for the control condition and least for the 360- and 180-msec conditions. Results are discussed in relation to the dynamics of the acoustic reflex, particularly reflex relaxation, reflex adaptation, and reflex temporal summation.

Tympanometric curves and otosclerosis.

Patients with clinical otosclerosis (28 ears) were examined audiometrically and with an electroacoustic impedance bridge. The results were compared to corresponding findings for normal ears. The tympanometric curves of surgically proven otosclerosis were found to differ characteristically from those of normal ears. A curve typical of footplate fixation shows a rapid increase in acoustic impedance as external auditory canal pressure is reduced from ambient air pressure.