Fitness of RNA virus decreased by Muller's ratchet

Why sex exists remains an unsolved problem in biology. If mutations are on the average deleterious, a high mutation rate can account for the evolution of sex. One form of this mutational hypothesis is Muller's ratchet. If the mutation rate is high, mutation-free individuals become rare and they can be lost by genetic drift in small populations. In asexual populations, as Muller noted, the loss is irreversible and the load of deleterious mutations increases in a ratchet-like manner with the successive loss of the least-mutated individuals. Sex can be advantageous because it increases the fitness of sexual populations by re-creating mutation-free individuals from mutated individuals and stops (or slows) Muller's ratchet. Although Muller's ratchet is an appealing hypothesis, it has been investigated and documented experimentally in only one group of organisms--ciliated protozoa. I initiated a study to examine the role of Muller's ratchet on the evolution of sex in RNA viruses and report here a significant decrease in fitness due to Muller's ratchet in 20 lineages of the RNA bacteriophage phi 6. These results show that deleterious mutations are generated at a sufficiently high rate to advance Muller's ratchet in an RNA virus and that beneficial, backward and compensatory mutations cannot stop the ratchet in the observed range of fitness decrease.     

Detection and characterization of a folding intermediate in barnase by NMR

Protein engineering is being developed for mapping the energetics and pathway of protein folding. From kinetic studies on wild-type and mutant proteins, the sequence and energetics of formation of tertiary interactions of side chains can be mapped and the formation of secondary structure inferred. Here we cross-check and complement results from this approach by using a recently developed procedure that traps and characterizes secondary structure in intermediate states using 1H NMR. The refolding of barnase is shown to be a multiphasic process in which the secondary structure in alpha-helices and beta-sheets and some turns is formed more rapidly than is the overall folding.     

High levels of unintegrated HIV-1 DNA in brain tissue of AIDS dementia patients

In the host cell, retroviral DNAs exist in three main forms: unintegrated linear, unintegrated circular, and integrated (the provirus). High levels of unintegrated forms of retroviral DNA often correlate with superinfection and accompanying cytopathic effects, as, for example, in the case of feline acquired immunodeficiency. In culture, HIV-1 infection also results in high levels of unintegrated viral DNA although direct correlations with cytopathicity have not been made. The low frequency of HIV-1-infected cells in patients has made it difficult to determine the structure of the viral DNA in fresh tissue samples from AIDS patients by standard methods such as Southern hybridization. The PCR technique however, which allows the detection of viral DNA at levels far below that possible by other hybridization methods is, in its conventional form, of limited use for quantitative analysis. To study the amount and form of HIV-1 DNA in primary tissue of AIDS patients we have therefore modified the PCR method. Our results indicate that each of the three species of viral DNA are detectable in blood and brain of AIDS patients, and that in autopsy samples from patients with HIV encephalitis there is a considerably higher proportion of unintegrated viral DNA.     

Molecular genetic basis of the histo-blood group ABO system

The histo-blood group ABO, the major human alloantigen system, involves three carbohydrate antigens (ABH). A, B and AB individuals express glycosyltransferase activities converting the H antigen into A or B antigens, whereas O(H) individuals lack such activity. Here we present a molecular basis for the ABO genotypes. The A and B genes differ in a few single-base substitutions, changing four amino-acid residues that may cause differences in A and B transferase specificity. A critical single-base deletion was found in the O gene, which results in an entirely different, inactive protein incapable of modifying the H antigen.     

Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death

The t(14; 18) chromosomal translocation of human follicular B-cell lymphoma juxtaposes the bcl-2 gene with the immunoglobulin heavy chain locus. The bcl-2 immunoglobulin fusion gene is markedly deregulated resulting in inappropriately elevated levels of bcl-2 RNA and protein. Transgenic mice bearing a bcl-2 immunoglobulin minigene demonstrate a polyclonal expansion of resting yet responsive IgM-IgD B cells which display prolonged cell survival but no increase in cell cycling. Moreover, deregulated bcl-2 extends the survival of certain haematopoietic cell lines following growth-factor deprivation. By using immunolocalization studies we now demonstrate that Bcl-2 is an integral inner mitochondrial membrane protein of relative molecular mass 25,000 (25k). Overexpression of Bcl-2 blocks the apoptotic death of a pro-B-lymphocyte cell line. Thus, Bcl-2 is unique among proto-oncogenes, being localized to mitochondria and interfering with programmed cell death independent of promoting cell division.     

Co-localization of molecules involved in antigen processing and presentation in an early endocytic compartment

The pathways of intracellular traffic involved in antigen processing and presentation have been defined by immunoelectron microscopy. The export pathway for class II histocompatibility molecules and the antigen import pathway meet in a peripheral endocytic compartment having all the molecular machinery believed to be required for antigen processing and presentation, including internalized surface immunoglobulins, proteolytic enzymes and invariant chains. This compartment defines a site where peptides from endocytosed antigen can bind class II molecules en route to the cell surface for presentation to T cells.     

No evidence for illegitimate young in monogamous and polygynous warblers

In animals with internal fertilization, paternity is uncertain. In birds, the occurrence of copulations outside the pair-bond has been documented in a number of species, but the extent to which these result in illegitimate young is largely unknown, and constitutes a major deficiency in our understanding of avian mating systems. The analysis of tandemly repeated sequences (minisatellites), has enhanced our ability to make individual identifications and paternity determinations. Here we describe the use of a bird minisatellite DNA probe in assigning paternity in natural populations of the monogamous willow warbler Phylloscopus trochilus and of the polygynous wood warbler Phylloscopus sibilatrix. In both species this probe detects a multiple locus pattern and a single locus that exhibits a variable number of tandem repeats. Although we observed intrusions by non-resident males into the territories of paired males and extra-pair copulations, no illegitimate offspring were detected among 176 young from 32 families of both species, implying that extra-pair copulations have little or no genetic impact.     

Structure of Arc repressor in solution: evidence for a family of beta-sheet DNA-binding proteins

The Arc repressor, which is involved in the switch between lysis and lysogeny of Salmonella bacteriophage P22, does not belong to any of the known classes of DNA-binding proteins. Mutagenesis studies show that the DNA-binding region is located in the 15 N-terminal amino-acid residues. We have now determined the three-dimensional structure of the Arc dimer from an extensive set of interproton-distance data obtained from 1H NMR spectroscopy. A priori, intra- and inter-monomer nuclear Overhauser effects (NOEs) cannot be distinguished for a symmetric dimer. But by using the homology with the Escherichia coli Met repressor we could interpret the NOEs unambiguously in an iterative structure refinement procedure. The final structure satisfies a large set of NOE constraints (1,352 for the dimer). It shows a strongly intertwined dimer, in which residues 8-14 of different monomers form an antiparallel beta-sheet. A model for the Arc repressor-operator complex can account for all available biochemical and genetic data. In this model two Arc dimers bind with their beta-sheet regions in successive major grooves on one side of the DNA helix, similar to the Met repressor interaction. Thus, Arc and Met repressors are members of the same family of proteins, which contain an antiparallel beta-sheet as the DNA-binding motif.     

Small G proteins are expressed ubiquitously in lymphoid cells and do not correspond to Bcl-2

The bcl-2 gene is consistently associated with t(14; 18) chromosomal translocations observed in a large fraction of human B-cell lymphomas. The t(14; 18) translocation results in deregulated expression of the bcl-2 gene and synthesis of inappropriately high levels of the Bcl-2 protein. Gene transfer studies suggest a role for Bcl-2 in cell survival, growth enhancement and oncogenic transformation. To test the suggestion that GTP-binding by Bcl-2 may mediate its biological effects we characterized the GTP-binding proteins in lymphoid cells expressing Bcl-2. Expression of several small GTP-binding proteins was found to be ubiquitous and did not vary with levels of Bcl-2. By using immunological, electrophoretic and cell-fractionation techniques, we separated Bcl-2 from G proteins of small relative molecular mass (Mr) and showed that it is incapable of binding GTP. Our results show that small Mr G proteins are widely expressed in lymphoid cells and that Bcl-2 is not a novel member of this GTP-binding protein family.