Influence of pH on the conformation and stability of mismatch base-pairs in DNA

A series of self-complementary dodecanucleotide duplexes containing two symmetrically disposed mismatches have been studied by pH-dependent, ultraviolet light melting techniques. The results indicate that A.C, and C.C mismatches are strongly stabilized by protonation and that the degree of stabilization of the A.C mismatch depends greatly on the flanking bases. In one case, a duplex containing two A.C mismatches is more stable than the native sequence below pH 5.5. The G.A mismatch displays conformational flexibility, with a protonated G(syn).A(anti) base-pair occurring in certain base stacking environments but not in others. The A.A and T.C mismatches are not stabilized at low pH. These solution studies correlate well with predictions based on X-ray crystallographic data.     

Characterization of an antibody-binding epitope from the 18-kDa protein on Mycobacterium leprae

A murine mAb, designated L5, appears to be specific for an epitope on a protein from Mycobacterium leprae of restricted distribution within the mycobacteria. This protein, of Mr 18,000 (18 kDa) is of interest because monoclonal antibodies raised against it do not appear to cross-react with other mycobacterial pathogens. The L5 antibody-binding epitope has been mapped by two complementary methods; expression of gene fragments and synthesis of short peptides. This L5-binding region of the 18-kDa protein (amino acids 109 to 115) shows some homology to a region of the GroEL heat shock family of proteins. Characterization of this antibody-binding epitope may lead to a reagent of use in early diagnosis of infection.     

Activation of tat-defective human immunodeficiency virus by ultraviolet light

Ultraviolet light (UV) is known to cause activation of gene expression from the human immunodeficiency virus type 1 (HIV-1) promoter. To address the question of whether tat-defective HIV-1 provirus could be rescued by UV irradiation we examined its effect on HeLa cells containing integrated proviruses with tat mutations. Exposure of these cells to an optimal dose of UV resulted in the production of infectious viruses. The degree of UV activation and reversion to infectious virus appeared to depend on the nature of the original tat mutation. Two of the mutants required cocultivation with tat-expressing cells to fully generate replication competent viruses, while a third mutant required only cocultivation with H9 cells. Sequencing of cDNA from cells infected with this last mutant demonstrated that the parental mutant sequence was retained and that genotypic revertants to the wild-type as well as new mutant sequences were generated. These results suggest that tat-defective HIV-1 provirus can be activated by UV and can subsequently revert to wild-type virus. This study raises the possibility that UV exposure of immune cells in the skin plays a role in the activation of defective HIV-1 in vivo.     

Microtubule Depolymerization Inhibits Ethanol-Induced Enhancement of GABAA Responses in Stably Transfected Cells

Abstract: We studied whether microtubule organization is important for actions of ethanol on GABA_A ergic responses by testing the effects of microtubule depolymerization on ethanol enhancement of GABA action in mouse L(tk⁻) cells stably transfected with GABA_A receptor α₁β₁γ_2L subunits. The microtubule-disrupting agents colchicine, taxol, and vinblastine completely blocked ethanol-induced enhancement of muscimol-stimulated chloride uptake. β-Lumicolchicine, a colchicine analogue that does not disrupt microtubules, had no effect on ethanol action. Colchicine did not alter the potentiating actions of flunitrazepam or pentobarbital on muscimol-stimulated chloride uptake. Thus, colchicine specifically inhibited the potentiating action of ethanol. From these findings, we conclude that intact microtubules are required for ethanol-induced enhancement of GABA_A responses and suggest that a mechanism involving microtubules produces posttranslational modifications that are necessary for ethanol sensitivity in this cell system.     

The activity of the high-affinity K+ uptake system Kdp sensitizes cells of Escherichia coli to methylglyoxal.

Expression of the Kdp system sensitizes cells to methylglyoxal (MG) whether this electrophile is added externally or is synthesized endogenously. The basis of this enhanced sensitivity is the maintenance of a higher cytoplasmic pH (pHi) in cells expressing Kdp. In such cells, MG elicits rapid cytoplasmic acidification via KefB and KefC, but the steady-state pHi attained is still too high to confer protection Lowering pHi further by incubation with acetate increases the sensitivity of cells to MG.     

‘Lymphocyte-like’cells in ascidians: Precursors for vertebrate lymphocytes?

There has been much speculation that the evolutionary precursors of vertebrate lymphocytes may exist in the ascidians (proto chordates), but conclusive evidence has remained elusive especially as membrane bound immunoglobulin has not been detected in any invertebrate. This paper reviews new evidence which indicates that the ‘lymphocyte-like’ cells in ascidians have functional, as well as morphological, similarities with vertebrate lymphocytes. Firstly, these cells have been linked with in vivo non-self recognition events such as allograft rejection in solitary ascidians and non-fusion reactions between colonial ascidians. Secondly, they have been shown to be cytotoxic towards xenogenic targets in vitro and to use cytolytic mechanisms similar to those of cytotoxic T-cells. Thirdly, they are able to proliferate in vitro in response to mitogens or allogeneic cells. It is therefore suggested that, apart from immunoglobulin production and clonal selection, there is persuasive evidence that the ‘lymphocyte-like’ cells of ascidians constitute a primordial form of vertebrate lymphocyte.     

Locus Heterogeneity for Waardenburg Syndrome is Predictive of Clinical Subtypes

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between −2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.     

Cloning of a higher-plant plastid omega-6 fatty acid desaturase cDNA and its expression in a cyanobacterium.

Oligomers based on amino acids conserved between known plant omega-3 and cyanobacterium omega-6 fatty acid desaturases were used to screen an Arabidopsis cDNA library for related sequences. An identified clone encoding a novel desaturase-like polypeptide was used to isolate its homologs from Glycine max and Brassica napus. The plant deduced amino acid sequences showed less than 27% similarity to known plant omega-6 and omega-3 desaturases but more than 48% similarity to cyanobacterial omega-6 desaturase, and they contained putative plastid transit sequences. Thus, we deduce that the plant cDNAs encode the plastid omega-6 desaturase. The identity was supported by expression of the B. napus cDNA in cyanobacterium. Synechococcus transformed with a chimeric gene that contains a prokaryotic promoter fused to the rapeseed cDNA encoding all but the first 73 amino acids partially converted its oleic acid fatty acid to linoleic acid, and the 16:1(9c) fatty acid was converted primarily to 16:2(9c, 12) in vivo. Thus, the plant omega-6 desaturase, which utilizes 16:1(7c) in plants, can utilize 16:1(9c) in the cyanobacterium. The plastid and cytosolic homologs of plant omega-6 desaturases are much more distantly related than those of omega-3 desaturases.     

Voltage-Gated Potassium Channel Genes Are Clustered in Paralogous Regions of the Mouse Genome

Cloning of the Drosophila Shaker gene established that a neurological phenotype including locomotor dysfunction can be caused by a mutation in a voltage-gated potassium (K) channel gene. Shaker sequences have been used to isolate a large family of related K channel genes from both flies and mammals. Toward elucidating the evolutionary relationship between loci and the potential causal connection that K channels may have to mammalian genetic disorders, we report here the genetic mapping of 12-16 different murine, voltage-gated K channel genes. We find that multiple genes, in some cases from distantly related K channel subfamilies, occur in clusters in the mouse genome. These mapping results suggest that the K channel gene subfamilies arose through ancient localized gene duplication events, followed by chromosomal duplications and rearrangements as well as further gene duplication. We also note that several neurologic disorders of both mouse and human are associated with the chromosomal regions containing K channel genes.     

Evolutionary epistemology as an overlapping,interlevel theory

I examine the branch of evolutionary epistemology which tries to account for the character of cognitive mechanisms in animals and humans by extending the biological theory of evolution to the neurophysiological substrates of cognition. Like Plotkin, I construe this branch as a struggling science, and attempt to characterize the sort of theory one might expect to find this truly interdisciplinary endeavor, an endeavor which encompasses not only evolutionary biology, cognitive psychology, and developmental neuroscience, but also and especially, the computational modeling of artificial life programming; I suggest that extending Schaffner's notion of interlevel theories to include both horizontal and vertical levels of abstraction best fits the theories currently being developed in cognitive science. Finally, I support this claim with examples drawn from computational modeling data using the genetic algorithm.