Multiple DNA and protein sequence alignment based on segment-to-segment comparison.

In this paper, a new way to think about, and to construct, pairwise as well as multiple alignments of DNA and protein sequences is proposed. Rather than forcing alignments to either align single residues or to introduce gaps by defining an alignment as a path running right from the source up to the sink in the associated dot-matrix diagram, we propose to consider alignments as consistent equivalence relations defined on the set of all positions occurring in all sequences under consideration. We also propose constructing alignments from whole segments exhibiting highly significant overall similarity rather than by aligning individual residues. Consequently, we present an alignment algorithm that (i) is based on segment-to-segment comparison instead of the commonly used residue-to-residue comparison and which (ii) avoids the well-known difficulties concerning the choice of appropriate gap penalties: gaps are not treated explicity, but remain as those parts of the sequences that do not belong to any of the aligned segments. Finally, we discuss the application of our algorithm to two test examples and compare it with commonly used alignment methods. As a first example, we aligned a set of 11 DNA sequences coding for functional helix-loop-helix proteins. Though the sequences show only low overall similarity, our program correctly aligned all of the 11 functional sites, which was a unique result among the methods tested. As a by-product, the reading frames of the sequences were identified. Next, we aligned a set of ribonuclease H proteins and compared our results with alignments produced by other programs as reported by McClure et al. [McClure, M. A., Vasi, T. K. & Fitch, W. M. (1994) Mol. Biol. Evol. 11, 571-592]. Our program was one of the best scoring programs. However, in contrast to other methods, our protein alignments are independent of user-defined parameters.

Deregulated expression of c-Myc in a translocation-negative plasmacytoma on extrachromosomal elements that carry IgH and myc genes

The induced expression of c-Myc in plasmacytomas in BALB/c mice is regularly associated with nonrandom chromosomal translocations that juxtapose the c-myc gene to one of the Ig loci on chromosome 12 (IgH), 6 (IgK), or 16 (IgL). The DCPC21 plasmacytoma belongs to a small group of plasmacytomas that are unusual in that they appear to be translocation-negative. In this paper, we show the absence of any c-myc-activating chromosomal translocation for the DCPC21 by using fluorescent in situ hybridization, chromosome painting, and spectral karyotyping. We find that DCPC21 harbors c-myc and IgH genes on extrachromosomal elements (EEs) from which c-myc is transcribed, as shown by c-myc mRNA tracks and extrachromosomal gene transfer experiments. The transcriptional activity of these EEs is supported further by the presence of the transcription-associated phosphorylation of histone H3 (H3P) on the EEs. Thus, our data suggest that in this plasmacytoma, c-Myc expression is achieved by an alternative mechanism. The expression of the c-Myc oncoprotein is initiated outside the chromosomal locations of the c-myc gene, i.e., from EEs, which can be considered functional genetic units. Our data also imply that other “translocation-negative” experimental and human tumors with fusion transcripts or oncogenic activation may indeed carry translocation(s), however, in an extrachromosomal form.

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1–1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)—this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.

Is blindsight like normal, near-threshold vision?

Blindsight is the rare and paradoxical ability of some human subjects with occipital lobe brain damage to discriminate unseen stimuli in their clinically blind field defects when forced-choice procedures are used, implying that lesions of striate cortex produce a sharp dissociation between visual performance and visual awareness. Skeptics have argued that this is no different from the behavior of normal subjects at the lower limits of conscious vision, at which such dissociations could arise trivially by using different response criteria during clinical and forced-choice tests. We tested this claim explicitly by measuring the sensitivity of a hemianopic patient independently of his response criterion in yes-no and forced-choice detection tasks with the same stimulus and found that, unlike normal controls, his sensitivity was significantly higher during the forced-choice task. Thus, the dissociation by which blindsight is defined is not simply due to a difference in the patients’ response bias between the two paradigms. This result implies that blindsight is unlike normal, near-threshold vision and that information about the stimulus is processed in blindsighted patients in an unusual way.

A JAK-STAT pathway regulates wing vein formation in Drosophila.

We present evidence that the JAK-STAT signal transduction pathway regulates multiple developmental processes in Drosophila. We screened for second-site mutations that suppress the phenotype of the hyperactive hopTum-1 Jak kinase, and recovered a mutation that meiotically maps to the known chromosomal position of D-Stat, a Drosophila stat gene. This hypomorphic mutation, termed statHJ contains a nucleotide substitution in the first D-Stat intron, resulting in a reduction in the number of correctly processed transcripts. Further, the abnormally processed mRNA encodes a truncated protein that has a dominant negative effect on transcriptional activation by the wild-type cDNA in cell culture. statHJ mutants exhibit patterning defects that include the formation of ectopic wing veins, similar to those seen in mutants of the epidermal growth factor/receptor pathway. Abnormalities in embryonic and adult segmentation and in tracheal development were also observed. The hopTum-1 and statHJ mutations can partially compensate for each other genetically, and Hop overexpression can increase D-Stat transcriptional activity in vitro, indicating that the gene products interact in a common regulatory pathway.